Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation.

• MeSH
• Cyclooxygenase 1 * MeSH
• DEAD Box Protein 58 MeSH
• DEAD-box RNA Helicases metabolism   genetics   MeSH
• Liver * metabolism   pathology   MeSH
• Humans MeSH
• Membrane Proteins MeSH
• Mitochondrial Proteins metabolism   genetics   MeSH
• Mitochondria metabolism   MeSH
• Mutation MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Oxidative Phosphorylation * MeSH
• Protein Biosynthesis MeSH
• Reactive Oxygen Species * metabolism   MeSH
• Electron Transport Complex IV * metabolism   genetics   MeSH
• RNA, Mitochondrial genetics   metabolism   MeSH
• Inflammation * metabolism   genetics   pathology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cyclooxygenase 1 * MeSH
• Ddx58 protein, mouse MeSH Browser
• DEAD Box Protein 58 MeSH
• DEAD-box RNA Helicases MeSH
• Membrane Proteins MeSH
• Mitochondrial Proteins MeSH
• Ptgs1 protein, mouse MeSH Browser
• Reactive Oxygen Species * MeSH
• Electron Transport Complex IV * MeSH
• RNA, Mitochondrial MeSH

Leber hereditary optic neuropathy is a primary mitochondrial disease characterized by acute visual loss due to the degeneration of retinal ganglion cells. In this study, we describe a patient carrying a rare missense heteroplasmic variant in MT-ND1, NC_012920.1:m.4135T>C (p.Tyr277His) manifesting with a typical bilateral painless decrease of the visual function, triggered by physical exercise or higher ambient temperature. Functional studies in muscle and fibroblasts show that amino acid substitution Tyr277 with His leads to only a negligibly decreased level of respiratory chain complex I (CI), but the formation of supercomplexes and the activity of the enzyme are disturbed noticeably. Our data indicate that although CI is successfully assembled in the patient's mitochondria, its function is hampered by the m.4135T>C variant, probably by stabilizing CI in its inactive form. We conclude that the m.4135T>C variant together with a combination of external factors is necessary to manifest the phenotype.

• Keywords
• complex I, mitochondria, mtDNA, optic neuropathy, supercomplexes,
• Publication type
• Journal Article MeSH
• Case Reports MeSH

Purpose: Retinal ischemia (RI) and progressive neuronal death are sight-threatening conditions. Mitochondrial (mt) dysfunction and fusion/fission processes have been suggested to play a role in the pathophysiology of RI. This study focuses on changes in the mt parameters of the neuroretina, retinal pigment epithelium (RPE) and choroid in a porcine high intraocular pressure (IOP)-induced RI minipig model. Methods: In one eye, an acute IOP elevation was induced in minipigs and compared to the other control eye. Activity and amount of respiratory chain complexes (RCC) were analyzed by spectrophotometry and Western blot, respectively. The coenzyme Q10 (CoQ10) content was measured using HPLC, and the ultrastructure of the mt was studied via transmission electron microscopy. The expression of selected mt-pathway genes was determined by RT-PCR. Results: At a functional level, increased RCC I activity and decreased total CoQ10 content were found in RPE cells. At a protein level, CORE2, a subunit of RCC III, and DRP1, was significantly decreased in the neuroretina. Drp1 and Opa1, protein-encoding genes responsible for mt quality control, were decreased in most of the samples from the RPE and neuroretina. Conclusions: The eyes of the minipig can be considered a potential RI model to study mt dysfunction in this disease. Strategies targeting mt protection may provide a promising way to delay the acute damage and onset of RI.

• Keywords
• coenzyme Q10, minipig, mitochondrial dysfunction, retinal ischemia,
• MeSH
• Glaucoma * metabolism   MeSH
• Ischemia metabolism   MeSH
• Carcinoma, Renal Cell * metabolism   MeSH
• Swine, Miniature MeSH
• Mitochondria metabolism   MeSH
• Kidney Neoplasms * metabolism   MeSH
• Intraocular Pressure MeSH
• Swine MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The Acyl-CoA-binding domain-containing protein (ACBD3) plays multiple roles across the cell. Although generally associated with the Golgi apparatus, it operates also in mitochondria. In steroidogenic cells, ACBD3 is an important part of a multiprotein complex transporting cholesterol into mitochondria. Balance in mitochondrial cholesterol is essential for proper mitochondrial protein biosynthesis, among others. We generated ACBD3 knock-out (ACBD3-KO) HEK293 and HeLa cells and characterized the impact of protein absence on mitochondria, Golgi, and lipid profile. In ACBD3-KO cells, cholesterol level and mitochondrial structure and functions are not altered, demonstrating that an alternative pathway of cholesterol transport into mitochondria exists. However, ACBD3-KO cells exhibit enlarged Golgi area with absence of stacks and ribbon-like formation, confirming the importance of ACBD3 in Golgi stacking. The glycosylation of the LAMP2 glycoprotein was not affected by the altered Golgi structure. Moreover, decreased sphingomyelins together with normal ceramides and sphingomyelin synthase activity reveal the importance of ACBD3 in ceramide transport from ER to Golgi.

• Keywords
• ACBD3, Golgi, OXPHOS, cholesterol, knock-out, mitochondria,
• MeSH
• Adaptor Proteins, Signal Transducing metabolism   MeSH
• Biological Transport physiology   MeSH
• Ceramides metabolism   MeSH
• Cholesterol metabolism   MeSH
• Glycosylation MeSH
• Golgi Apparatus metabolism   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Humans MeSH
• Membrane Proteins metabolism   MeSH
• Lysosomal-Associated Membrane Protein 2 metabolism   MeSH
• Mitochondria metabolism   MeSH
• Signal Transduction physiology   MeSH
• Transferases (Other Substituted Phosphate Groups) metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• ACBD3 protein, human MeSH Browser
• Adaptor Proteins, Signal Transducing MeSH
• Ceramides MeSH
• Cholesterol MeSH
• Membrane Proteins MeSH
• Lysosomal-Associated Membrane Protein 2 MeSH
• phosphatidylcholine-ceramide phosphocholine transferase MeSH Browser
• Transferases (Other Substituted Phosphate Groups) MeSH

The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI.

• Keywords
• COX, COX4, OXPHOS, biogenesis interdependency, cI, cIV, cIV assembly, complex I, complexome profiling, knock-out, mitochondria, mitochondrial protein synthesis,
• MeSH
• Glycolysis MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mitochondrial Diseases metabolism   MeSH
• Mitochondrial Proteins biosynthesis   MeSH
• Oxidative Phosphorylation MeSH
• Protein Subunits metabolism   MeSH
• Protein Biosynthesis * MeSH
• Electron Transport Complex IV metabolism   MeSH
• Oxygen Consumption MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• COX4I1 protein, human MeSH Browser
• Mitochondrial Proteins MeSH
• Protein Subunits MeSH
• Electron Transport Complex IV MeSH

Mitochondrial protein quality control is crucial for the maintenance of correct mitochondrial homeostasis. It is ensured by several specific mitochondrial proteases located across the various mitochondrial subcompartments. Here, we focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 (AFG3 Like Matrix AAA Peptidase Subunit 2) and YME1L (YME1 like ATPase) of mitochondrial inner membrane AAA (ATPases Associated with diverse cellular Activities) complexes in the maintenance of mitochondrial structure and respiratory chain integrity. We demonstrate that loss of AFG3L2 and YME1L, both alone and in combination, results in diminished cell proliferation, fragmentation of mitochondrial reticulum, altered cristae morphogenesis, and defective respiratory chain biogenesis. The double AFG3L2/YME1L knockdown cells showed marked upregulation of OPA1 protein forms, with the most prominent increase in short OPA1 (optic atrophy 1). Loss of either protease led to marked elevation in OMA1 (OMA1 zinc metallopeptidase) (60 kDa) and severe reduction in the SPG7 (paraplegin) subunit of the m-AAA complex. Loss of the YME1L subunit led to an increased Drp1 level in mitochondrial fractions. While loss of YME1L impaired biogenesis and function of complex I, knockdown of AFG3L2 mainly affected the assembly and function of complex IV. Our results suggest cooperative and partly redundant functions of AFG3L2 and YME1L in the maintenance of mitochondrial structure and respiratory chain biogenesis and stress the importance of correct proteostasis for mitochondrial integrity.

• Keywords
• AAA complex, AFG3L2, YME1L, mitochondria, protease,
• MeSH
• ATPases Associated with Diverse Cellular Activities genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Metalloendopeptidases genetics   metabolism   MeSH
• Mitochondrial Membranes metabolism   MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Mitochondria metabolism   ultrastructure   MeSH
• Cell Proliferation genetics   physiology   MeSH
• ATP-Dependent Proteases genetics   metabolism   MeSH
• Microscopy, Electron, Transmission MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• AFG3L2 protein, human MeSH Browser
• ATPases Associated with Diverse Cellular Activities MeSH
• Metalloendopeptidases MeSH
• Mitochondrial Proteins MeSH
• ATP-Dependent Proteases MeSH
• YME1L1 protein, human MeSH Browser

p53 is a major cellular tumor suppressor that in addition to its nuclear, transcription-dependent activity is also known to function extranuclearly. Cellular stressors such as reactive oxygen species can promote translocation of p53 into mitochondria where it acts to protect mitochondrial genome or trigger cell death via transcription-independent manner. Here we report that the mammalian homologue of yeast mitochondrial Afg1 ATPase (LACE1) promotes translocation of p53 into mitochondria. We further show that LACE1 exhibits significant pro-apoptotic activity, which is dependent on p53, and that the protein is required for normal mitochondrial respiratory function. LACE1 physically interacts with p53 and is necessary for mitomycin c-induced translocation of p53 into mitochondria. Conversely, increased expression of LACE1 partitions p53 to mitochondria, causes reduction in nuclear p53 content and induces apoptosis. Thus, LACE1 mediates mitochondrial translocation of p53 and its transcription-independent apoptosis.

• Keywords
• LACE1, apoptosis, mitochondria, p53, translocation,
• MeSH
• Adenosine Triphosphatases metabolism   MeSH
• Apoptosis physiology   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria metabolism   MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• Transfection MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• AFG1L protein, human MeSH Browser
• Mitochondrial Proteins MeSH
• Tumor Suppressor Protein p53 MeSH
• TP53 protein, human MeSH Browser

Mitochondrial protein SURF1 is a specific assembly factor of cytochrome c oxidase (COX), but its function is poorly understood. SURF1 gene mutations cause a severe COX deficiency manifesting as the Leigh syndrome in humans, whereas in mice SURF1(-/-) knockout leads only to a mild COX defect. We used SURF1(-/-) mouse model for detailed analysis of disturbed COX assembly and COX ability to incorporate into respiratory supercomplexes (SCs) in different tissues and fibroblasts. Furthermore, we compared fibroblasts from SURF1(-/-) mouse and SURF1 patients to reveal interspecies differences in kinetics of COX biogenesis using 2D electrophoresis, immunodetection, arrest of mitochondrial proteosynthesis and pulse-chase metabolic labeling. The crucial differences observed are an accumulation of abundant COX1 assembly intermediates, low content of COX monomer and preferential recruitment of COX into I-III2-IVn SCs in SURF1 patient fibroblasts, whereas SURF1(-/-) mouse fibroblasts were characterized by low content of COX1 assembly intermediates and milder decrease in COX monomer, which appeared more stable. This pattern was even less pronounced in SURF1(-/-) mouse liver and brain. Both the control and SURF1(-/-) mice revealed only negligible formation of the I-III2-IVn SCs and marked tissue differences in the contents of COX dimer and III2-IV SCs, also less noticeable in liver and brain than in heart and muscle. Our studies support the view that COX assembly is much more dependent on SURF1 in humans than in mice. We also demonstrate markedly lower ability of mouse COX to form I-III2-IVn supercomplexes, pointing to tissue-specific and species-specific differences in COX biogenesis.

• Keywords
• Cytochrome c oxidase, Doxycycline, Leigh syndrome, Pulse-chase, Respiratory supercomplexes, SURF1(−/−) mouse knockout,
• MeSH
• Species Specificity MeSH
• Fibroblasts metabolism   pathology   MeSH
• Leigh Disease genetics   metabolism   pathology   MeSH
• Humans MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Organ Specificity MeSH
• Electron Transport Complex IV genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• Mitochondrial Proteins MeSH
• Electron Transport Complex IV MeSH
• Surf-1 protein MeSH Browser

Mitochondrial disorders are caused by defects in mitochondrial or nuclear DNA. Although the existence of large deletions in mitochondrial DNA (mtDNA) is well known, deletions affecting whole genes are not commonly described in patients with mitochondrial disorders. Based on the results of whole-genome analyses, copy number variations (CNVs) occur frequently in the human genome and may overlap with many genes associated with clinical phenotypes. We report the discovery of two large heterozygous CNVs on 22q13.33 in two patients with mitochondrial disorders. The first patient harboured a novel point mutation c.667G>A (p.D223N) in the SCO2 gene in combination with a paternally inherited 87-kb deletion. As hypertrophic cardiomyopathy (HCMP) was not documented in the patient, this observation prompted us to compare his clinical features with all 44 reported SCO2 patients in the literature. Surprisingly, the review shows that HCMP was present in only about 50% of the SCO2 patients with non-neonatal onset. In the second patient, who had mitochondrial neurogastrointestinal encephalopathy (MNGIE), a maternally inherited 175-kb deletion and the paternally inherited point mutation c.261G>T (p.E87D) in the TYMP gene were identified.

• MeSH
• Point Mutation * MeSH
• Child MeSH
• Cardiomyopathy, Hypertrophic, Familial diagnosis   genetics   MeSH
• Infant MeSH
• Humans MeSH
• Chromosomes, Human, Pair 22 genetics   MeSH
• Mitochondrial Encephalomyopathies diagnosis   genetics   MeSH
• Mitochondrial Proteins genetics   MeSH
• Molecular Chaperones MeSH
• Ophthalmoplegia congenital   MeSH
• Intestinal Pseudo-Obstruction diagnosis   genetics   MeSH
• Muscular Dystrophy, Oculopharyngeal MeSH
• Thymidine Phosphorylase genetics   MeSH
• Carrier Proteins genetics   MeSH
• DNA Copy Number Variations * MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Mitochondrial Proteins MeSH
• Molecular Chaperones MeSH
• SCO2 protein, human MeSH Browser
• Thymidine Phosphorylase MeSH
• Carrier Proteins MeSH
• TYMP protein, human MeSH Browser

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i-AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.

• MeSH
• Apoptosis MeSH
• ATPases Associated with Diverse Cellular Activities MeSH
• Gene Knockdown Techniques MeSH
• GTP Phosphohydrolases metabolism   MeSH
• Humans MeSH
• Metalloendopeptidases metabolism   MeSH
• Mitochondrial Membranes metabolism   MeSH
• Mitochondrial Proteins MeSH
• Mitochondria metabolism   MeSH
• NADH, NADPH Oxidoreductases metabolism   MeSH
• Cell Proliferation * MeSH
• ATP-Dependent Proteases metabolism   MeSH
• Peptide Hydrolases metabolism   MeSH
• Protein Isoforms metabolism   MeSH
• Electron Transport Complex I MeSH
• Electron Transport Complex IV metabolism   MeSH
• Saccharomyces cerevisiae Proteins metabolism   MeSH
• Saccharomyces cerevisiae cytology   metabolism   MeSH
• Electron Transport * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATPases Associated with Diverse Cellular Activities MeSH
• GTP Phosphohydrolases MeSH
• Metalloendopeptidases MeSH
• Mitochondrial Proteins MeSH
• NADH, NADPH Oxidoreductases MeSH
• NDUFB6 protein, human MeSH Browser
• OPA1 protein, human MeSH Browser
• ATP-Dependent Proteases MeSH
• Peptide Hydrolases MeSH
• Protein Isoforms MeSH
• Electron Transport Complex I MeSH
• Electron Transport Complex IV MeSH
• Saccharomyces cerevisiae Proteins MeSH
• YME1 protein, S cerevisiae MeSH Browser
• YME1L1 protein, human MeSH Browser