Human mitochondrial complex I assembly: A dynamic and versatile process

One can but admire the intricate way in which biomolecular structures are formed and cooperate to allow proper cellular function. A prominent example of such intricacy is the assembly of the five inner membrane embedded enzymatic complexes of the mitochondrial oxidative phosphorylation (OXPHOS) system, which involves the stepwise combination of > 80 subunits and prosthetic groups encoded by both the mitochondrial and nuclear genomes. This review will focus on the assembly of the most complicated OXPHOS structure: complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3). Recent studies into complex I assembly in human cells have resulted in several models elucidating a thus far enigmatic process. In this review, special attention will be given to the overlap between the various assembly models proposed in different organisms. Complex I being a complicated structure, its assembly must be prone to some form of coordination. This is where chaperone proteins come into play, some of which may relate complex I assembly to processes such as apoptosis and even immunity.     

Understanding mitochondrial complex I assembly in health and disease

Complex I (NADH:ubiquinone oxidoreductase) is the largest multimeric enzyme complex of the mitochondrial respiratory chain, which is responsible for electron transport and the generation of a proton gradient across the mitochondrial inner membrane to drive ATP production. Eukaryotic complex I consists of 14 conserved subunits, which are homologous to the bacterial subunits, and more than 26 accessory subunits. In mammals, complex I consists of 45 subunits, which must be assembled correctly to form the properly functioning mature complex. Complex I dysfunction is the most common oxidative phosphorylation (OXPHOS) disorder in humans and defects in the complex I assembly process are often observed. This assembly process has been difficult to characterize because of its large size, the lack of a high resolution structure for complex I, and its dual control by nuclear and mitochondrial DNA. However, in recent years, some of the atomic structure of the complex has been resolved and new insights into complex I assembly have been generated. Furthermore, a number of proteins have been identified as assembly factors for complex I biogenesis and many patients carrying mutations in genes associated with complex I deficiency and mitochondrial diseases have been discovered. Here, we review the current knowledge of the eukaryotic complex I assembly process and new insights from the identification of novel assembly factors. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

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.     

TMEM70 functions in the assembly of complexes I and V

Protein complexes from the oxidative phosphorylation (OXPHOS) system are assembled with the help of proteins called assembly factors. We here delineate the function of the inner mitochondrial membrane protein TMEM70, in which mutations have been linked to OXPHOS deficiencies, using a combination of BioID, complexome profiling and coevolution analyses. TMEM70 interacts with complex I and V and for both complexes the loss of TMEM70 results in the accumulation of an assembly intermediate followed by a reduction of the next assembly intermediate in the pathway. This indicates that TMEM70 has a role in the stability of membrane-bound subassemblies or in the membrane recruitment of subunits into the forming complex. Independent evidence for a role of TMEM70 in OXPHOS assembly comes from evolutionary analyses. The TMEM70/TMEM186/TMEM223 protein family, of which we show that TMEM186 and TMEM223 are mitochondrial in human as well, only occurs in species with OXPHOS complexes. Our results validate the use of combining complexome profiling with BioID and evolutionary analyses in elucidating congenital defects in protein complex assembly.     

Molecular base of biochemical complex I deficiency

The oxidative phosphorylation (OXPHOS) system, consisting of five enzyme complexes (I-V) together with 2 electron carriers, has an important role in the energy metabolism of the cell. With 45 subunits, complex I is the first and largest complex of the respiratory chain. It is under bigenomic control and a proper interaction between the mitochondrial and the nuclear genome is important for a good biogenesis and functioning of the complex. Isolated complex I deficiency is the most frequently diagnosed form of mitochondrial disorders caused by the disturbance of the OXPHOS system. It has a wide clinical variety and, at present, in many patients the underlying genetic cause of the complex I deficiency is still not known. In this review, the role of complex I in the oxidative phosphorylation and the localization and function of the different complex I subunits will be described. Furthermore, a brief overview of the assembly process and biochemical studies, performed when a patient is suspected of a mitochondrial disorder is given. Finally, the present knowledge for molecular base of complex I deficiency is described and the findings in a research cohort of patients with complex I deficiency are reported. Identifying new genes encoding proteins involved in complex I biogenesis is challenging and in the near future new powerful techniques will make high throughput screening possible. Progress in elucidating the genetic defect causing complex I deficiencies is important for a better genetic counseling, prenatal diagnostic possibilities and further development of new treatment strategies to cure the complex I deficiencies in the future.     

Structures of mitochondrial oxidative phosphorylation supercomplexes and mechanisms for their stabilisation

Oxidative phosphorylation (OXPHOS) is the main source of energy in eukaryotic cells. This process is performed by means of electron flow between four enzymes, of which three are proton pumps, in the inner mitochondrial membrane. The energy accumulated in the proton gradient over the inner membrane is utilized for ATP synthesis by a fifth OXPHOS complex, ATP synthase. Four of the OXPHOS protein complexes associate into stable entities called respiratory supercomplexes. This review summarises the current view on the arrangement of the electron transport chain in mitochondrial cristae. The functional role of the supramolecular organisation of the OXPHOS system and the factors that stabilise such organisation are highlighted. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

GRIM-19, a cell death regulatory protein, is essential for assembly and function of mitochondrial complex I

Mitochondria play essential roles in cellular energy production via the oxidative phosphorylation system (OXPHOS) consisting of five multiprotein complexes and also in the initiation of apoptosis. NADH:ubiquinone oxidoreductase (complex I) is the largest complex that catalyzes the first step of electron transfer in the OXPHOS system. GRIM-19 was originally identified as a nuclear protein with apoptotic nature in interferon (IFN)- and all-trans-retinoic acid (RA)-induced tumor cells. To reveal its biological role, we generated mice deficient in GRIM-19 by gene targeting. Homologous deletion of GRIM-19 causes embryonic lethality at embryonic day 9.5. GRIM-19(-/-) blastocysts show retarded growth in vitro and, strikingly, display abnormal mitochondrial structure, morphology, and cellular distribution. We reexamined the cellular localization of GRIM-19 in various cell types and found its primary localization in the mitochondria. Furthermore, GRIM-19 is detected in the native form of mitochondrial complex I. Finally, we show that elimination of GRIM-19 destroys the assembly and electron transfer activity of complex I and also influences the other complexes in the mitochondrial respiratory chain. Our result demonstrates that GRIM-19, a gene product with a specific role in IFN-RA-induced cell death, is a functional component of mitochondrial complex I and is essential for early embryonic development.     

Enhanced liver but not muscle OXPHOS in diabetes and reduced glucose output by complex I inhibition

Mitochondrial function is critical in energy metabolism. To fully capture how the mitochondrial function changes in metabolic disorders, we investigated mitochondrial function in liver and muscle of animal models mimicking different types and stages of diabetes. Type 1 diabetic mice were induced by streptozotocin (STZ) injection. The db/db mice were used as type 2 diabetic model. High-fat diet-induced obese mice represented pre-diabetic stage of type 2 diabetes. Oxidative phosphorylation (OXPHOS) of isolated mitochondria was measured with Clark-type oxygen electrode. Both in early and late stages of type 1 diabetes, liver mitochondrial OXPHOS increased markedly with complex IV-dependent OXPHOS being the most prominent. However, ATP, ADP and AMP contents in the tissue did not change. In pre-diabetes and early stage of type 2 diabetes, liver mitochondrial complex I and II-dependent OXPHOS increased greatly then declined to almost normal at late stage of type 2 diabetes, among which alteration of complex I-dependent OXPHOS was the most significant. In contrast, muscle mitochondrial OXPHOS in HFD, early-stage type 1 and 2 diabetic mice, did not change. In vitro, among inhibitors to each complex, only complex I inhibitor rotenone decreased glucose output in primary hepatocytes without cytotoxicity both in the absence and presence of oleic acid (OA). Rotenone affected cellular energy state and had no effects on cellular and mitochondrial reactive oxygen species production. Taken together, the mitochondrial OXPHOS of liver but not muscle increased in obesity and diabetes, and only complex I inhibition may ameliorate hyperglycaemia via lowering hepatic glucose production.     

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).     

Eukaryotic complex I: functional diversity and experimental systems to unravel the assembly process

With more than 40 subunits, one FMN co-factor and eight FeS clusters, complex I or NADH:ubiquinone oxidoreductase is the largest multimeric respiratory enzyme in the mitochondria. In this review, we focus on the diversity of eukaryotic complex I. We describe the additional activities that have been reported to be associated with mitochondrial complex I and discuss their physiological significance. The recent identification of complex I-like enzymes in the hydrogenosome, a mitochondria-derived organelle is also discussed here. Complex I assembly in the mitochondrial inner membrane is an intricate process that requires the cooperation of the nuclear and mitochondrial genomes. The most prevalent forms of mitochondrial dysfunction in humans are deficiencies in complex I and remarkably, the molecular basis for 60% of complex I-linked defects is currently unknown. This suggests that mutations in yet-to-be-discovered assembly genes should exist. We review the different experimental systems for the study of complex I assembly. To our knowledge, in none of them, large screenings of complex I mutants have been performed. We propose that the unicellular green alga Chlamydomonas reinhardtii is a promising system for such a study. Complex I mutants can be easily scored on a phenotypical basis and a large number of transformants generated by insertional mutagenesis can be screened, which opens the possibility to find new genes involved in the assembly of the enzyme. Moreover, mitochondrial transformation, a recent technological advance, is now available, allowing the manipulation of all five complex I mitochondrial genes in this organism.     

Expression of Rattus norvegicus mtDNA in Mus musculus cells results in multiple respiratory chain defects

The production of in vitro and in vivo models of mitochondrial DNA (mtDNA) defects is currently limited by a lack of characterized mouse cell mtDNA mutants that may be expected to model human mitochondrial diseases. Here we describe the creation of transmitochondrial mouse (Mus musculus) cells repopulated with mtDNA from different murid species (xenomitochondrial cybrids). The closely related Mus spretus mtDNA is readily maintained when introduced into M. musculus mtDNA-less (rho(0)) cells, and the resulting cybrids have normal oxidative phosphorylation (OXPHOS). When the more distantly related Rattus norvegicus mtDNA is transferred to the mouse nuclear background the mtDNA is replicated, transcribed, and translated efficiently. However, function of several OXPHOS complexes that depend on the coordinated assembly of nuclear and mtDNA-encoded proteins is impaired. Complex I activity in the Rattus xenocybrid was 46% of the control mean; complex III was 37%, and complex IV was 78%. These defects combined to restrict maximal respiration to 12-31% of the control and M. spretus xenocybrids, as measured polarographically using isolated cybrid mitochondria. These defects are distinct to those previously reported for human/primate xenocybrids. It should be possible to produce other mouse xenocybrid constructs with less severe OXPHOS phenotypes, to model human mtDNA diseases.     

Screening for active small molecules in mitochondrial complex I deficient patient's fibroblasts, reveals AICAR as the most beneficial compound

Congenital deficiency of the mitochondrial respiratory chain complex I (CI) is a common defect of oxidative phosphorylation (OXPHOS). Despite major advances in the biochemical and molecular diagnostics and the deciphering of CI structure, function assembly and pathomechanism, there is currently no satisfactory cure for patients with mitochondrial complex I defects. Small molecules provide one feasible therapeutic option, however their use has not been systematically evaluated using a standardized experimental system. In order to evaluate potentially therapeutic compounds, we set up a relatively simple system measuring different parameters using only a small amount of patient's fibroblasts, in glucose free medium, where growth is highly OXPOS dependent. Ten different compounds were screened using fibroblasts derived from seven CI patients, harboring different mutations.5-Aminoimidazole-4-carboxamide ribotide (AICAR) was found to be the most beneficial compound improving growth and ATP content while decreasing ROS production. AICAR also increased mitochondrial biogenesis without altering mitochondrial membrane potential (Δψ). Fluorescence microscopy data supported increased mitochondrial biogenesis and activation of the AMP activated protein kinase (AMPK). Other compounds such as; bezafibrate and oltipraz were rated as favorable while polyphenolic phytochemicals (resverastrol, grape seed extract, genistein and epigallocatechin gallate) were found not significant or detrimental. Although the results have to be verified by more thorough investigation of additional OXPHOS parameters, preliminary rapid screening of potential therapeutic compounds in individual patient's fibroblasts could direct and advance personalized medical treatment.     

The role of mitochondrial respiration in physiological and evolutionary adaptation

Aerobic mitochondria serve as the power sources of eukaryotes by producing ATP through oxidative phosphorylation (OXPHOS). The enzymes involved in OXPHOS are multisubunit complexes encoded by both nuclear and mitochondrial DNA. Thus, regulation of respiration is necessarily a highly coordinated process that must organize production, assembly and function of mitochondria to meet an organism's energetic needs. Here I review the role of OXPHOS in metabolic adaptation and diversification of higher animals. On a physiological timescale, endocrine-initiated signaling pathways allow organisms to modulate respiratory enzyme concentration and function under changing environmental conditions. On an evolutionary timescale, mitochondrial enzymes are targets of natural selection, balancing cytonuclear coevolutionary constraints against physiological innovation. By synthesizing our knowledge of biochemistry, physiology and evolution of respiratory regulation, I propose that we can now explore questions at the interface of these fields, from molecular translation of environmental cues to selection on mitochondrial haplotype variation.     

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.     

Mutations in human nuclear genes encoding for subunits of mitochondrial respiratory complex I: the NDUFS4 gene

Among the mitochondrial disorders, complex I deficiencies are encountered frequently. Although some complex I deficiencies have been associated with mitochondrial DNA mutations, in the majority of the complex I-deficient patients mutations of nuclear genes are expected. This review attempts to summarize genetic defects affecting nuclear encoded subunits of complex I reported to date focusing on those found in the NDUFS4 gene. NDUFS4 product is 18 kDa protein which appears to have a dual role in complex I, at least: cAMP-dependent phosphorylation activates the complex; non-sense mutation of NDUFS4 prevents normal assembly of a functional complex in the inner mitochondrial membrane.     

Energetic requirements and bioenergetic modulation of mitochondrial morphology and dynamics

Mitochondria are the site where oxidative phosphorylations (OXPHOSs) take place. Fusion and fission reactions allow them to change their overall morphology, which ranges from networks of elongated and branched filaments to collections of small individual organelles. It is assumed that mitochondrial bioenergetics and dynamics are linked and that mitochondrial morphology reflects their functional status. This review shows that the links between mitochondrial dynamics and bioenergetics are complex and that mitochondrial deficiencies are not systematically associated to fragmentation. In mammals, mitochondrial fragmentation is observed upon inhibition of OXPHOS with drugs, but not in most cellular models with OXPHOS deficits of genetic origin. In yeast, mitochondrial biogenesis and filament interconnectivity augment with increasing respiratory capacity, but mutation or inhibition of the respiratory chain does not provoke major morphological changes. Significant structural and morphological alterations appear restricted to mutation of genes involved in assembly or function of the F₁F₀-ATP-synthase. Finally, ex vivo studies (in mammals) and in vitro studies (in yeast) confirm the essential role of the inner membrane potential for mitochondrial fusion.     

线粒体电子传递系统的组装及其生物学意义

电子传递链亦称呼吸链,由位于线粒体内膜的I、II、III、IV 4种复合物组成,负责电子传递和产生质子梯度。电子主要从复合物I进入电子传递链,经复合物III传递至复合物IV。电子传递系统的组装是一个十分复杂的过程,目前已知主要有约69个结构亚基以及至少16个组装因子参与了人类复合物I、III、IV的组装,这些蛋白质由核基因组与线粒体基因组共同编码。对线粒体电子传递系统的蛋白质组成及其结构已研究得较为清楚,但对它们的组装了解得还比较初步。许多人类线粒体疾病是由于电子传递系统的功能障碍引起的,其中又有许多是由于该系统中一个或多个部件的错误组装引起的。研究这些缺陷不仅能够加深对线粒体疾病发病机理的了解,也有助于揭示线粒体功能的调控机制。将着重对电子传递系统复合物的组装及其与人类疾病关系的研究进展进行综述。     

Supercomplex organization of the oxidative phosphorylation enzymes in yeast mitochondria

Accumulating evidence indicates that the enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) are co-assembled into higher-ordered supercomplexes within the mitochondrial inner membrane. This review will focus largely on the OXPHOS supercomplexes of the yeast Saccharomyces cerevisiae. The recent evidence to indicate that diversity in the populations of the cytochrome bc (1)-COX supercomplexes exist shall be outlined. In addition, the existence of dimeric/oligomeric F(1)F(o)-ATP synthase complexes and their proposed role in establishment of the cristae architecture of the inner mitochondrial membrane shall also be discussed.     

The protein import and assembly machinery of the mitochondrial outer membrane

The process of mitochondrial protein import has been studied for many years. Despite this attention, many processes associated with mitochondrial biogenesis are poorly understood. Insight into one of these processes, assembly of beta-barrel proteins into the mitochondrial outer membrane, will be discussed. This review focuses on recent data that suggest that assembly of beta-barrel proteins into the outer mitochondrial membrane is dependent on a newly identified protein complex termed the sorting and assembly machinery (SAM complex). Members of the SAM complex have been identified in both eukaryotic and prokaryotic organisms, suggesting that the process of beta-barrel assembly into membranes has been conserved through evolution.     

The impact of mitochondrial tRNA mutations on the amount of ATP synthase differs in the brain compared to other tissues

The impact of point mutations in mitochondrial tRNA genes on the amount and stability of respiratory chain complexes and ATP synthase (OXPHOS) has been broadly characterized in cultured skin fibroblasts, skeletal muscle samples, and mitochondrial cybrids. However, less is known about how these mutations affect other tissues, especially the brain. We have compared OXPHOS protein deficiency patterns in skeletal muscle mitochondria of patients with Leigh (8363G>A), MERRF (8344A>G), and MELAS (3243A>G) syndromes. Both mutations that affect mt-tRNA(Lys) (8363G>A, 8344A>G) resulted in severe combined deficiency of complexes I and IV, compared to an isolated severe defect of complex I in the 3243A>G sample (mt-tRNA(LeuUUR). Furthermore, we compared obtained patterns with those found in the heart, frontal cortex, and liver of 8363G>A and 3243A>G patients. In the frontal cortex mitochondria of both patients, the patterns of OXPHOS deficiencies differed substantially from those observed in other tissues, and this difference was particularly striking for ATP synthase. Surprisingly, in the frontal cortex of the 3243A>G patient, whose ATP synthase level was below the detection limit, the assembly of complex IV, as inferred from 2D-PAGE immunoblotting, appeared to be hindered by some factor other than the availability of mtDNA-encoded subunits.