Subunits of Mitochondrial Complex I Exist as Part of Matrix- and Membrane-associated Subcomplexes in Living Cells*S⃞

Mitochondrial complex I (CI) is a large assembly of 45 different subunits, and defects in its biogenesis are the most frequent cause of mitochondrial disorders. In vitro evidence suggests a stepwise assembly process involving pre-assembled modules. However, whether these modules also exist in vivo is as yet unresolved. To answer this question, we here applied submitochondrial fluorescence recovery after photobleaching to HEK293 cells expressing 6 GFP-tagged subunits selected on the basis of current CI assembly models. We established that each subunit was partially present in a virtually immobile fraction, possibly representing the holo-enzyme. Four subunits (NDUFV1, NDUFV2, NDUFA2, and NDUFA12) were also present as highly mobile matrix-soluble monomers, whereas, in sharp contrast, the other two subunits (NDUFB6 and NDUFS3) were additionally present in a slowly mobile fraction. In the case of the integral membrane protein NDUFB6, this fraction most likely represented one or more membrane-bound subassemblies, whereas biochemical evidence suggested that for the NDUFS3 protein this fraction most probably corresponded to a matrix-soluble subassembly. Our results provide first time evidence for the existence of CI subassemblies in mitochondria of living cells

Cytosolic signaling protein Ecsit also localizes to mitochondria where it interacts with chaperone NDUFAF1 and functions in complex I assembly

Ecsit is a cytosolic adaptor protein essential for inflammatory response and embryonic development via the Toll-like and BMP (bone morphogenetic protein) signal transduction pathways, respectively. Here, we demonstrate a mitochondrial function for Ecsit (an evolutionary conserved signaling intermediate in Toll pathways) in the assembly of mitochondrial complex I (NADH:ubiquinone oxidoreductase). An N-terminal targeting signal directs Ecsit to mitochondria, where it interacts with assembly chaperone NDUFAF1 in 500- to 850-kDa complexes as demonstrated by affinity purification and vice versa RNA interference (RNAi) knockdowns. In addition, Ecsit knockdown results in severely impaired complex I assembly and disturbed mitochondrial function. These findings support a function for Ecsit in the assembly or stability of mitochondrial complex I, possibly linking assembly of oxidative phosphorylation complexes to inflammatory response and embryonic development

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

Assembly factors as a new class of disease genes for mitochondrial complex I deficiency: cause, pathology and treatment options.

Item does not contain fulltextComplex I deficiency is the most frequent cause of oxidative phosphorylation disorders. The disease features a large diversity of clinical symptoms often leading to progressive encephalomyopathies with a fatal outcome. There is currently no cure, and although disease-causing mutations have been found in the genes encoding complex I subunits, half of the cases remain unexplained. However, in the past 5 years a new class of complex I disease genes has emerged with the finding of specific assembly factors. So far nine such genes have been described and it is believed that in the near future more will be found. In this review, we will address whether the functions of these chaperones point towards a general molecular mechanism of disease and whether this enables us to design a treatment for complex I deficiency.1 januari 201

Acyl-CoA dehydrogenase 9 is required for the biogenesis of oxidative phosphorylation complex I.

Contains fulltext : 87919.pdf (publisher's version ) (Closed access)Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified member of the acyl-CoA dehydrogenase family. It closely resembles very long-chain acyl-CoA dehydrogenase (VLCAD), involved in mitochondrial beta oxidation of long-chain fatty acids. Contrary to its previously proposed involvement in fatty acid oxidation, we describe a role for ACAD9 in oxidative phosphorylation. ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required for the assembly of complex I. Furthermore, ACAD9 mutations result in complex I deficiency and not in disturbed long-chain fatty acid oxidation. This strongly contrasts with its evolutionary ancestor VLCAD, which we show is not required for complex I assembly and clearly plays a role in fatty acid oxidation. Our results demonstrate that two closely related metabolic enzymes have diverged at the root of the vertebrate lineage to function in two separate mitochondrial metabolic pathways and have clinical implications for the diagnosis of complex I deficiency

LC-MS/MS data processing and protein profile generation.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068340#pone-0068340-g002" target="_blank"><b>Figure 2A</b></a> provides an overview for the main steps in LC-MS/MS data processing. The acquired mass spectrometry data is used to identify peptides and protein for each gel slice via database searches using the Mascot search engine. Resulting peptide identifications together with the LC-MS data are used as input for the label-free quantitation by the Ideal-Q software that integrates the chromatographic peak surfaces for each peptide from respective extracted ion currents. Quantitative information from all LC-MS/MS analyses is then used to determine the relative abundance for each peptide in every gel slice. The final step in data processing uses the peptide profiles to reconstruct the migration profile for each individual protein in the blue native gel separation. Details for the reconstruction of the protein migration profiles are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068340#pone-0068340-g002" target="_blank"><b>figure 2B</b></a> with data for the cytochrome <i>c</i> oxidase subunit 6 C protein. First, all peptide profiles of a protein are used to generate a similarity matrix that contains the Pearson's correlation coefficients between each peptide profile. This information is then used to calculate a similarity score for each peptide which is defined as the sum of all the Pearson's correlation coefficients for the peptide from the similarity matrix. The next step uses a Grubb's outlier test on the calculated similarity scores to discard peptide profiles that poorly correspond with the general peptide migration profile. Finally, the peptide migration profiles are ranked in descending order of their similarity score of which the 5 highest scoring peptide profiles are used to construct the protein migration profile by averaging.</p

Representative hierarchical clustering results of mitochondrial protein profiles.

<p>Profiles from proteins with mitochondrial annotation (MITOP2 reference dataset or Gene Ontology) or mitochondrial prediction (MITOP2 SVM) were subjected to hierarchical clustering (uncentered Pearson's correlation metric, optimized leaf order, and complete linkage distance). Five representative clusters are shown that contain the five mitochondrial oxidative phosphorylation system (OXPHOS) complexes. Clusters that are referenced in the text are indicated in the HCL tree with numbers that correspond with cluster numbers defined by the MEV software.</p