Erika Fernandez-Vizarra

Supercomplex Assembly Determines Electron Flux in the Mitochondrial Electron Transport Chain

Respiration Refined Cells derive energy from redox reactions mediated by mitochondrial enzymes that form the electron transport chain. The enzymes can form large complexes, known as supercomplexes, whose function has been controversial. Lapuente-Brun et al. (p. 1567) discovered that a mouse protein, supercomplex assembly factor I (SCAFI), specifically modulates assembly of respiratory complexes into supercomplexes. Formation of the supercomplexes appears to cause electrons to be processed differently, depending on the substrate from which they are derived. Ordered formation of supercomplexes of respiratory enzymes influences metabolic efficiency in response to food supply. The textbook description of mitochondrial respiratory complexes (RCs) views them as free-moving entities linked by the mobile carriers coenzyme Q (CoQ) and cytochrome c (cyt c). This model (known as the fluid model) is challenged by the proposal that all RCs except complex II can associate in supercomplexes (SCs). The proposed SCs are the respirasome (complexes I, III, and IV), complexes I and III, and complexes III and IV. The role of SCs is unclear, and their existence is debated. By genetic modulation of interactions between complexes I and III and III and IV, we show that these associations define dedicated CoQ and cyt c pools and that SC assembly is dynamic and organizes electron flux to optimize the use of available substrates.

MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion

The mitochondrial (mt) DNA depletion syndromes (MDDS) are genetic disorders characterized by a severe, tissue-specific decrease of mtDNA copy number, leading to organ failure. There are two main clinical presentations: myopathic (OMIM 609560) and hepatocerebral (OMIM 251880). Known mutant genes, including TK2 (ref. 2), SUCLA2 (ref. 3), DGUOK (ref. 4) and POLG, account for only a fraction of MDDS cases. We found a new locus for hepatocerebral MDDS on chromosome 2p21-23 and prioritized the genes on this locus using a new integrative genomics strategy. One of the top-scoring candidates was the human ortholog of the mouse kidney disease gene Mpv17 (ref. 8). We found disease-segregating mutations in three families with hepatocerebral MDDS and demonstrated that, contrary to the alleged peroxisomal localization of the MPV17 gene product, MPV17 is a mitochondrial inner membrane protein, and its absence or malfunction causes oxidative phosphorylation (OXPHOS) failure and mtDNA depletion, not only in affected individuals but also in Mpv17−/− mice.

Assembly of the oxidative phosphorylation system in humans: what we have learned by studying its defects.

Assembly of the oxidative phosphorylation (OXPHOS) system in the mitochondrial inner membrane is an intricate process in which many factors must interact. The OXPHOS system is composed of four respiratory chain complexes, which are responsible for electron transport and generation of the proton gradient in the mitochondrial intermembrane space, and of the ATP synthase that uses this proton gradient to produce ATP. Mitochondrial human disorders are caused by dysfunction of the OXPHOS system, and many of them are associated with altered assembly of one or more components of the OXPHOS system. The study of assembly defects in patients has been useful in unraveling and/or gaining a complete understanding of the processes by which these large multimeric complexes are formed. We review here current knowledge of the biogenesis of OXPHOS complexes based on investigation of the corresponding disorders.

Infantile Encephalopathy and Defective Mitochondrial DNA Translation in Patients with Mutations of Mitochondrial Elongation Factors EFG1 and EFTu

Mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial DNA (mtDNA)-encoded RNAs and nuclear DNA-encoded proteins. Although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in RNA-encoding mtDNA genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. Genetic investigation involving patients with defective mitochondrial translation led us to the discovery of novel mutations in the mitochondrial elongation factor G1 (EFG1) in one affected baby and, for the first time, in the mitochondrial elongation factor Tu (EFTu) in another one. Both patients were affected by severe lactic acidosis and rapidly progressive, fatal encephalopathy. The EFG1-mutant patient had early-onset Leigh syndrome, whereas the EFTu-mutant patient had severe infantile macrocystic leukodystrophy with micropolygyria. Structural modeling enabled us to make predictions about the effects of the mutations at the molecular level. Yeast and mammalian cell systems proved the pathogenic role of the mutant alleles by functional complementation in vivo. Nuclear-gene abnormalities causing mitochondrial translation defects represent a new, potentially broad field of mitochondrial medicine. Investigation of these defects is important to expand the molecular characterization of mitochondrial disorders and also may contribute to the elucidation of the complex control mechanisms, which regulate this fundamental pathway of mtDNA homeostasis.

Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells.

This article describes a quick basic method adapted for the purification of mammalian mitochondria from different sources. The organelles obtained using this protocol are suitable for the investigation of biogenetic activities such as enzyme activity, mtDNA, mtRNA, mitochondrial protein synthesis, and mitochondrial tRNA aminoacylation. In addition, these mitochondria are capable of efficient protein import and the investigation of mtDNA/protein interactions by DNA footprinting is also possible.

Severe Infantile Encephalomyopathy Caused by a Mutation in COX6B1, a Nucleus-Encoded Subunit of Cytochrome C Oxidase

Cytochrome c oxidase (COX) deficiency, one of the most common respiratory-chain defects in humans, has been associated with mutations in either mitochondrial DNA genes or nucleus-encoded proteins that are not part in but promote the biogenesis of COX. Mutations of nucleus-encoded structural subunits were sought for but never found in COX-defective patients, leading to the conjecture that they may be incompatible with extra-uterine survival. We report a disease-associated mutation in one such subunit, COX6B1. Nuclear-encoded COX genes should be reconsidered and included in the diagnostic mutational screening of human disorders related to COX deficiency.

Loss of mitochondrial protease OMA1 alters processing of the GTPase OPA1 and causes obesity and defective thermogenesis in mice

Mitochondria are dynamic subcellular organelles that convert nutrient intermediates into readily available energy equivalents. Optimal mitochondrial function is ensured by a highly evolved quality control system, coordinated by protein machinery that regulates a process of continual fusion and fission. In this work, we provide in vivo evidence that the ATP‐independent metalloprotease OMA1 plays an essential role in the proteolytic inactivation of the dynamin‐related GTPase OPA1 (optic atrophy 1). We also show that OMA1 deficiency causes a profound perturbation of the mitochondrial fusion–fission equilibrium that has important implications for metabolic homeostasis. Thus, ablation of OMA1 in mice results in marked transcriptional changes in genes of lipid and glucose metabolic pathways and substantial alterations in circulating blood parameters. Additionally, Oma1‐mutant mice exhibit an increase in body weight due to increased adipose mass, hepatic steatosis, decreased energy expenditure and impaired thermogenenesis. These alterations are especially significant under metabolic stress conditions, indicating that an intact OMA1‐OPA1 system is essential for developing the appropriate adaptive response to different metabolic stressors such as a high‐fat diet or cold‐shock. This study provides the first description of an unexpected role in energy metabolism for the metalloprotease OMA1 and reinforces the importance of mitochondrial quality control for normal metabolic function.

Isolation of mitochondria for biogenetical studies: An update

The use of good quality preparations of isolated mitochondria is necessary when studying the mitochondrial biogenetical activities. This article explains a fast and simple method for the purification of mammalian mitochondria from different tissues and cultured cells, that is suitable for the analysis of many aspects of the organelles biogenesis. The mitochondria isolated following the protocol described here, are highly active and capable of DNA, RNA and protein synthesis. Mitochondrial tRNA aminoacylation, mtDNA-protein interactions and specific import of added proteins into the organelles, can also be studied using this kind of preparations.

Early-onset liver mtDNA depletion and late-onset proteinuric nephropathy in Mpv17 knockout mice

In humans, MPV17 mutations are responsible for severe mitochondrial depletion syndrome, mainly affecting the liver and the nervous system. To gain insight into physiopathology of MPV17-related disease, we investigated an available Mpv17 knockout animal model. We found severe mtDNA depletion in liver and, albeit to a lesser extent, in skeletal muscle, whereas hardly any depletion was detected in brain and kidney, up to 1 year after birth. Mouse embryonic fibroblasts did show mtDNA depletion, but only after several culturing passages, or in a serumless culturing medium. In spite of severe mtDNA depletion, only moderate decrease in respiratory chain enzymatic activities, and mild cytoarchitectural alterations, were observed in the Mpv17−/− livers, but neither cirrhosis nor failure ever occurred in this organ at any age. The mtDNA transcription rate was markedly increased in liver, which could contribute to compensate the severe mtDNA depletion. This phenomenon was associated with specific downregulation of Mterf1, a negative modulator of mtDNA transcription. The most relevant clinical features involved skin, inner ear and kidney. The coat of the Mpv17−/− mice turned gray early in adulthood, and 18-month or older mice developed focal segmental glomerulosclerosis (FSGS) with massive proteinuria. Concomitant degeneration of cochlear sensory epithelia was reported as well. These symptoms were associated with significantly shorter lifespan. Coincidental with the onset of FSGS, there was hardly any mtDNA left in the glomerular tufts. These results demonstrate that Mpv17 controls mtDNA copy number by a highly tissue- and possibly cytotype-specific mechanism.

FASTKD2 Nonsense Mutation in an Infantile Mitochondrial Encephalomyopathy Associated with Cytochrome C Oxidase Deficiency

In two siblings we found a mitochondrial encephalomyopathy, characterized by developmental delay, hemiplegia, convulsions, asymmetrical brain atrophy, and low cytochrome c oxidase (COX) activity in skeletal muscle. The disease locus was identified on chromosome 2 by homozygosity mapping; candidate genes were prioritized for their known or predicted mitochondrial localization and then sequenced in probands and controls. A homozygous nonsense mutation in the KIAA0971 gene segregated with the disease in the proband family. The corresponding protein is known as fas activated serine-threonine kinase domain 2, FASTKD2. Confocal immunofluorescence colocalized a tagged recombinant FASTKD2 protein with mitochondrial markers, and membrane-potential-dependent in vitro mitochondrial import was demonstrated in isolated mitochondria. In staurosporine-induced-apoptosis experiments, decreased nuclear fragmentation was detected in treated mutant versus control fibroblasts. In conclusion, we found a loss-of-function mutation in a gene segregating with a peculiar mitochondrial encephalomyopathy associated with COX deficiency in skeletal muscle. The corresponding protein is localized in the mitochondrial inner compartment. Preliminary data indicate that FASTKD2 plays a role in mitochondrial apoptosis.