Mutations in NDUFAF3 (C3ORF60), Encoding an NDUFAF4 (C6ORF66)-Interacting Complex I Assembly Protein, Cause Fatal Neonatal Mitochondrial Disease

Mitochondrial complex I deficiency is the most prevalent and least understood disorder of the oxidative phosphorylation system. The genetic cause of many cases of isolated complex I deficiency is unknown because of insufficient understanding of the complex I assembly process and the factors involved. We performed homozygosity mapping and gene sequencing to identify the genetic defect in five complex I-deficient patients from three different families. All patients harbored mutations in the NDUFAF3 (C3ORF60) gene, of which the pathogenic nature was assessed by NDUFAF3-GFP baculovirus complementation in fibroblasts. We found that NDUFAF3 is a genuine mitochondrial complex I assembly protein that interacts with complex I subunits. Furthermore, we show that NDUFAF3 tightly interacts with NDUFAF4 (C6ORF66), a protein previously implicated in complex I deficiency. Additional gene conservation analysis links NDUFAF3 to bacterial-membrane-insertion gene cluster SecF/SecD/YajC and to C8ORF38, also implicated in complex I deficiency. These data not only show that NDUFAF3 mutations cause complex I deficiency but also relate different complex I disease genes by the close cooperation of their encoded proteins during the assembly process.     

Novel role for mitochondria: protein kinase Ctheta-dependent oxidative signaling organelles in activation-induced T-cell death

Reactive oxygen species (ROS) play a key role in regulation of activation-induced T-cell death (AICD) by induction of CD95L expression. However, the molecular source and the signaling steps necessary for ROS production are largely unknown. Here, we show that the proximal T-cell receptor-signaling machinery, including ZAP70 (zeta chain-associated protein kinase 70), LAT (linker of activated T cells), SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), PLCgamma1 (phospholipase Cgamma1), and PKCtheta (protein kinase Ctheta), are crucial for ROS production. PKCtheta is translocated to the mitochondria. By using cells depleted of mitochondrial DNA, we identified the mitochondria as the source of activation-induced ROS. Inhibition of mitochondrial electron transport complex I assembly by small interfering RNA (siRNA)-mediated knockdown of the chaperone NDUFAF1 resulted in a block of ROS production. Complex I-derived ROS are converted into a hydrogen peroxide signal by the mitochondrial superoxide dismutase. This signal is essential for CD95L expression, as inhibition of complex I assembly by NDUFAF1-specific siRNA prevents AICD. Similar results were obtained when metformin, an antidiabetic drug and mild complex I inhibitor, was used. Thus, we demonstrate for the first time that PKCtheta-dependent ROS generation by mitochondrial complex I is essential for AICD.     

Identification and functional analysis of mitochondrial complex I assembly factor homologues in C. elegans

The biogenesis of mitochondrial NADH:ubiquinone oxidoreductase (complex I) requires several assembly chaperones. These so-called complex I assembly factors have emerged as a new class of human disease genes. Here, we identified putative assembly factor homologues in Caenorhabditis elegans. We demonstrate that two candidates (C50B8.3/NUAF-1, homologue of NDUFAF1 and R07H5.3/NUAF-3, homologue of NDUFAF3) clearly affect complex I function. Assembly factor deficient worms were shorter, showed a diminished brood size and displayed reduced fat content. Our results suggest that mitochondrial complex I biogenesis is evolutionarily conserved. Moreover, Caenorhabditis elegans appears to be a promising model organism to study assembly factor related human diseases.     

Human CIA30 is involved in the early assembly of mitochondrial complex I and mutations in its gene cause disease

In humans, complex I of the respiratory chain is composed of seven mitochondrial DNA (mtDNA)-encoded and 38 nuclear-encoded subunits that assemble together in a process that is poorly defined. To date, only two complex I assembly factors have been identified and how each functions is not clear. Here, we show that the human complex I assembly factor CIA30 (complex I intermediate associated protein) associates with newly translated mtDNA-encoded complex I subunits at early stages in their assembly before dissociating at a later stage. Using antibodies we identified a CIA30-deficient patient who presented with cardioencephalomyopathy and reduced levels and activity of complex I. Genetic analysis revealed the patient had mutations in both alleles of the NDUFAF1 gene that encodes CIA30. Complex I assembly in patient cells was defective at early stages with subunits being degraded. Complementing the deficiency in patient fibroblasts with normal CIA30 using a novel lentiviral system restored steady-state complex I levels. Our results indicate that CIA30 is a crucial component in the early assembly of complex I and mutations in its gene can cause mitochondrial disease.     

Novel Insights into the Role of Neurospora crassa NDUFAF2, an Evolutionarily Conserved Mitochondrial Complex I Assembly Factor

Complex I deficiency is commonly associated with mitochondrial oxidative phosphorylation diseases. Mutations in nuclear genes encoding structural subunits or assembly factors of complex I have been increasingly identified as the cause of the diseases. One such factor, NDUFAF2, is a paralog of the NDUFA12 structural subunit of the enzyme, but the mechanism by which it exerts its function remains unknown. Herein, we demonstrate that the Neurospora crassa NDUFAF2 homologue, the 13.4L protein, is a late assembly factor that associates with complex I assembly intermediates containing the membrane arm and the connecting part but lacking the N module of the enzyme. Furthermore, we provide evidence that dissociation of the assembly factor is dependent on the incorporation of the putative regulatory module composed of the subunits of 13.4 (NDUFA12), 18.4 (NDUFS6), and 21 (NDUFS4) kDa. Our results demonstrate that the 13.4L protein is a complex I assembly factor functionally conserved from fungi to mammals.     

Defective mitochondrial translation differently affects the live cell dynamics of complex I subunits

Complex I (CI) of the oxidative phosphorylation system is assembled from 45 subunits encoded by both the mitochondrial and nuclear DNA. Defective mitochondrial translation is a major cause of mitochondrial disorders and proper understanding of its mechanisms and consequences is fundamental to rational treatment design. Here, we used a live cell approach to assess its consequences on CI assembly. The approach consisted of fluorescence recovery after photobleaching (FRAP) imaging of the effect of mitochondrial translation inhibition by chloramphenicol (CAP) on the dynamics of AcGFP1-tagged CI subunits NDUFV1, NDUFS3, NDUFA2 and NDUFB6 and assembly factor NDUFAF4. CAP increased the mobile fraction of the subunits, but not NDUFAF4, and decreased the amount of CI, demonstrating that CI is relatively immobile and does not associate with NDUFAF4. CAP increased the recovery kinetics of NDUFV1-AcGFP1 to the same value as obtained with AcGFP1 alone, indicative of the removal of unbound NDUFV1 from the mitochondrial matrix. Conversely, CAP decreased the mobility of NDUFS3-AcGFP1 and, to a lesser extent, NDUFB6-AcGFP1, suggestive of their enrichment in less mobile subassemblies. Little, if any, change in mobility of NDUFA2-AcGFP1 could be detected, suggesting that the dynamics of this accessory subunit of the matrix arm remains unaltered. Finally, CAP increased the mobility of NDUFAF4-AcGFP1, indicative of interaction with a more mobile membrane-bound subassembly. Our results show that the protein interactions of CI subunits and assembly factors are differently altered when mitochondrial translation is defective.     

A forward genetic screen identifies mutants deficient for mitochondrial complex I assembly in Chlamydomonas reinhardtii

Mitochondrial complex I is the largest multimeric enzyme of the respiratory chain. The lack of a model system with facile genetics has limited the molecular dissection of complex I assembly. Using Chlamydomonas reinhardtii as an experimental system to screen for complex I defects, we isolated, via forward genetics, amc1-7 nuclear mutants (for assembly of mitochondrial complex I) displaying reduced or no complex I activity. Blue native (BN)-PAGE and immunoblot analyses revealed that amc3 and amc4 accumulate reduced levels of the complex I holoenzyme (950 kDa) while all other amc mutants fail to accumulate a mature complex. In amc1, -2, -5-7, the detection of a 700 kDa subcomplex retaining NADH dehydrogenase activity indicates an arrest in the assembly process. Genetic analyses established that amc5 and amc7 are alleles of the same locus while amc1-4 and amc6 define distinct complementation groups. The locus defined by the amc5 and amc7 alleles corresponds to the NUOB10 gene, encoding PDSW, a subunit of the membrane arm of complex I. This is the first report of a forward genetic screen yielding the isolation of complex I mutants. This work illustrates the potential of using Chlamydomonas as a genetically tractable organism to decipher complex I manufacture.     

In vivo chlorophyll fluorescence screening allows the isolation of a Chlamydomonas mutant defective for NDUFAF3, an assembly factor involved in mitochondrial complex I assembly

The qualitative screening method used to select complex I mutants in the microalga Chlamydomonas, based on reduced growth under heterotrophic conditions, is not suitable for high‐throughput screening. In order to develop a fast screening method based on measurements of chlorophyll fluorescence, we first demonstrated that complex I mutants displayed decreased photosystem II efficiency in the genetic background of a photosynthetic mutation leading to reduced formation of the electrochemical proton gradient in the chloroplast (pgrl1 mutation). In contrast, single mutants (complex I and pgrl1 mutants) could not be distinguished from the wild type by their photosystem II efficiency under the conditions tested. We next performed insertional mutagenesis on the pgrl1 mutant. Out of about 3000 hygromycin‐resistant insertional transformants, 46 had decreased photosystem II efficiency and three were complex I mutants. One of the mutants was tagged and whole genome sequencing identified the resistance cassette in NDUFAF3, a homolog of the human NDUFAF3 gene, encoding for an assembly factor involved in complex I assembly. Complemented strains showed restored complex I activity and assembly. Overall, we describe here a screening method which is fast and particularly suited for the identification of Chlamydomonas complex I mutants.     

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.     

CIA30 complex I assembly factor: a candidate for human complex I deficiency?

The human mitochondrial NADH:ubiquinone oxidoreductase (complex I), the first complex of the oxidative phosphorylation system, is composed of at least 42 subunits. Little is known about the assembly process of these subunits into the mature complex. Recently, two proteins in Neurospora crassa have been found to be involved in the assembly of complex I. These proteins are not constituent parts of the mature complex but are associated with smaller intermediate complexes of the assembly process and have a chaperone-like function. We have characterized the human homologue of one of these two complex I intermediate associated proteins, named CIA30, and show that expression of the human CIA30 protein is ubiquitous with a slightly higher expression in various heart tissues, kidney, lung and liver. As deletion of the Neurospora crassa CIA genes results in severe disruption of the assembly process, human CIA30 can be considered as a candidate gene related to complex I deficiency. Thirteen patients with an isolated complex I deficiency, but who were ruled out for mutations in the 35 nuclear genes of the complex and mtDNA, were subjected to mutational analysis of the gene coding for the human CIA30 protein. Four new single nucleotide polymorphisms (SNPs) were detected but no functional mutation was found.     

Role of the conserved cysteine residues of the 11.5 kDa subunit in complex I catalytic properties

Mitochondrial complex I exists as a mixture of two inter-convertible forms: active (A) and de-activated (D), the latter being sensitive to SH-modifying compounds. To investigate if the conserved cysteine-rich 11.5 kDa subunit of Neurospora crassa complex I is involved in this process, we subjected the corresponding genomic DNA to site-directed mutagenesis. The four cysteine residues of the subunit were separately substituted with serine residues and the resulting proteins were independently expressed in a null-mutant strain. All of the obtained mutant strains were able to assemble a complex I with similar kinetic properties to those observed in the wild-type enzyme, indicating that none of the cysteine residues of the 11.5 kDa protein is individually relevant for the A/D transition process. Diminished amounts of assembled complex I seem to be the major effect of these specific mutations. The cysteine residues are likely important to the acquisition and stabilization of the correct 11.5 kDa protein conformation and this is reflected in the assembly/stability of complex I.     

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).     

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.     

Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6

Background

The quaternary structure of eukaryotic NADH:ubiquinone oxidoreductase (complex I), the largest complex of the oxidative phosphorylation, is still mostly unresolved. Furthermore, it is unknown where transiently bound assembly factors interact with complex I. We therefore asked whether the evolution of complex I contains information about its 3D topology and the binding positions of its assembly factors. We approached these questions by correlating the evolutionary rates of eukaryotic complex I subunits using the mirror-tree method and mapping the results into a 3D representation by multidimensional scaling.

Results

More than 60% of the evolutionary correlation among the conserved seven subunits of the complex I matrix arm can be explained by the physical distance between the subunits. The three-dimensional evolutionary model of the eukaryotic conserved matrix arm has a striking similarity to the matrix arm quaternary structure in the bacterium Thermus thermophilus (rmsd=19 ?) and supports the previous finding that in eukaryotes the N-module is turned relative to the Q-module when compared to bacteria. By contrast, the evolutionary rates contained little information about the structure of the membrane arm. A large evolutionary model of 45 subunits and assembly factors allows to predict subunit positions and interactions (rmsd = 52.6 ?). The model supports an interaction of NDUFAF3, C8orf38 and C2orf56 during the assembly of the proximal matrix arm and the membrane arm. The model further suggests a tight relationship between the assembly factor NUBPL and NDUFA2, which both have been linked to iron-sulfur cluster assembly, as well as between NDUFA12 and its paralog, the assembly factor NDUFAF2.

Conclusions

The physical distance between subunits of complex I is a major correlate of the rate of protein evolution in the complex I matrix arm and is sufficient to infer parts of the complex??s structure with high accuracy. The resulting evolutionary model predicts the positions of a number of subunits and assembly factors.     

Impact of mutations affecting ND mitochondria-encoded subunits on the activity and assembly of complex I in Chlamydomonas. Implication for the structural organization of the enzyme

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) comprises more than 35 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components (ND1, ND2, ND4, ND5 and ND6) are coded for by the mitochondrial genome. Here, we characterize two mitochondrial mutants (dum5 and dum17) showing strong reduction or inactivation of complex I activity: dum5 is a 1T deletion in the 3' UTR of nd5 whereas dum17 is a 1T deletion in the coding sequence of nd6. The impact of these mutations and of mutations affecting nd1, nd4 and nd4/nd5 genes on the assembly of complex I is investigated. After separation of the respiratory complexes by blue native (BN)-PAGE or sucrose gradient centrifugation, we demonstrate that the absence of intact ND1 or ND6 subunit prevents the assembly of the 850 kDa whole complex, whereas the loss of ND4 or ND4/ND5 leads to the formation of a subcomplex of 650 kDa present in reduced amount. The implications of our findings for the possible role of these ND subunits on the activity of complex I and for the structural organization of the membrane arm of the enzyme are discussed. In mitochondria from all the strains analyzed, we moreover detected a 160-210 kDa fragment comprising the hydrophilic 49 kDa and 76 kDa subunits of the complex I peripheral arm and showing NADH dehydrogenase activity.     

Identification of mitochondrial complex I assembly intermediates by tracing tagged NDUFS3 demonstrates the entry point of mitochondrial subunits

Biogenesis of human mitochondrial complex I (CI) requires the coordinated assembly of 45 subunits derived from both the mitochondrial and nuclear genome. The presence of CI subcomplexes in CI-deficient cells suggests that assembly occurs in distinct steps. However, discriminating between products of assembly or instability is problematic. Using an inducible NDUFS3-green fluorescent protein (GFP) expression system in HEK293 cells, we here provide direct evidence for the stepwise assembly of CI. Upon induction, six distinct NDUFS3-GFP-containing subcomplexes gradually appeared on a blue native Western blot also observed in wild type HEK293 mitochondria. Their stability was demonstrated by differential solubilization and heat incubation, which additionally allowed their distinction from specific products of CI instability and breakdown. Inhibition of mitochondrial translation under conditions of steady state labeling resulted in an accumulation of two of the NDUFS3-GFP-containing subcomplexes (100 and 150 kDa) and concomitant disappearance of the fully assembled complex. Lifting inhibition reversed this effect, demonstrating that these two subcomplexes are true assembly intermediates. Composition analysis showed that this event was accompanied by the incorporation of at least one mitochondrial DNA-encoded subunit, thereby revealing the first entry point of these subunits.     

Knockdown of human Oxa1l impairs the biogenesis of F1Fo-ATP synthase and NADH:ubiquinone oxidoreductase

The Oxa1 protein is a founding member of the evolutionarily conserved Oxa1/Alb3/YidC protein family, which is involved in the biogenesis of membrane proteins in mitochondria, chloroplasts and bacteria. The predicted human homologue, Oxa1l, was originally identified by partial functional complementation of the respiratory growth defect of the yeast oxa1 mutant. Here we demonstrate that both the endogenous human Oxa1l, with an apparent molecular mass of 42 kDa, and the Oxa1l-FLAG chimeric protein localize exclusively to mitochondria in HEK293 cells. Furthermore, human Oxa1l was found to be an integral membrane protein, and, using two-dimensional blue native/denaturing PAGE, the majority of the protein was identified as part of a 600-700 kDa complex. The stable short hairpin (sh)RNA-mediated knockdown of Oxa1l in HEK293 cells resulted in markedly decreased steady-state levels and ATP hydrolytic activity of the F₁F_o-ATP synthase and moderately reduced levels and activity of NADH:ubiquinone oxidoreductase (complex I). However, no significant accumulation of corresponding sub-complexes could be detected on blue native immunoblots. Intriguingly, the achieved depletion of Oxa1l protein did not adversely affect the assembly or activity of cytochrome c oxidase or the cytochrome bc₁ complex. Taken together, our results indicate that human Oxa1l represents a mitochondrial integral membrane protein required for the correct biogenesis of F₁F_o-ATP synthase and NADH:ubiquinone oxidoreductase.     

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。     

Expression of Yeast NDI1 Rescues a Drosophila Complex I Assembly Defect

Defects in mitochondrial electron transport chain (ETC) function have been implicated in a number of neurodegenerative disorders, cancer, and aging. Mitochondrial complex I (NADH dehydrogenase) is the largest and most complicated enzyme of the ETC with 45 subunits originating from two separate genomes. The biogenesis of complex I is an intricate process that requires multiple steps, subassemblies, and assembly factors. Here, we report the generation and characterization of a Drosophila model of complex I assembly factor deficiency. We show that CG7598 (dCIA30), the Drosophila homolog of human complex I assembly factor Ndufaf1, is necessary for proper complex I assembly. Reduced expression of dCIA30 results in the loss of the complex I holoenzyme band in blue-native polyacrylamide gel electrophoresis and loss of NADH:ubiquinone oxidoreductase activity in isolated mitochondria. The complex I assembly defect, caused by mutation or RNAi of dCIA30, has repercussions both during development and adulthood in Drosophila, including developmental arrest at the pupal stage and reduced stress resistance during adulthood. Expression of the single-subunit yeast alternative NADH dehydrogenase, Ndi1, can partially or wholly rescue phenotypes associated with the complex I assembly defect. Our work shows that CG7598/dCIA30 is a functional homolog of Ndufaf1 and adds to the accumulating evidence that transgenic NDI1 expression is a viable therapy for disorders arising from complex I deficiency.