RESUMO
The prokaryotic translation elongation factor P (EF-P) and the eukaryotic/archaeal counterparts eIF5A/aIF5A are proteins that serve a crucial role in mitigating ribosomal stalling during the translation of specific sequences, notably those containing consecutive proline residues (1,2). Although mitochondrial DNA-encoded proteins synthesized by mitochondrial ribosomes also contain polyproline stretches, an EF-P/eIF5A mitochondrial counterpart remains unidentified. Here, we show that the missing factor is TACO1, a protein causative of a juvenile form of neurodegenerative Leigh's syndrome associated with cytochrome c oxidase deficiency, until now believed to be a translational activator of COX1 mRNA. By using a combination of metabolic labeling, puromycin release and mitoribosome profiling experiments, we show that TACO1 is required for the rapid synthesis of the polyproline-rich COX1 and COX3 cytochrome c oxidase subunits, while its requirement is negligible for other mitochondrial DNA-encoded proteins. In agreement with a role in translation efficiency regulation, we show that TACO1 cooperates with the N-terminal extension of the large ribosomal subunit bL27m to provide stability to the peptidyl-transferase center during elongation. This study illuminates the translation elongation dynamics within human mitochondria, a TACO1-mediated biological mechanism in place to mitigate mitoribosome stalling at polyproline stretches during protein synthesis, and the pathological implications of its malfunction.
RESUMO
The human mitochondrial ribosome contains three [2Fe-2S] clusters whose assembly pathway, role, and implications for mitochondrial and metabolic diseases are unknown. Here, structure-function correlation studies show that the clusters play a structural role during mitoribosome assembly. To uncover the assembly pathway, we have examined the effect of silencing the expression of Fe-S cluster biosynthetic and delivery factors on mitoribosome stability. We find that the mitoribosome receives its [2Fe-2S] clusters from the GLRX5-BOLA3 node. Additionally, the assembly of the small subunit depends on the mitoribosome biogenesis factor METTL17, recently reported containing a [4Fe-4S] cluster, which we propose is inserted via the ISCA1-NFU1 node. Consistently, fibroblasts from subjects suffering from 'multiple mitochondrial dysfunction' syndrome due to mutations in BOLA3 or NFU1 display previously unrecognized attenuation of mitochondrial protein synthesis that contributes to their cellular and pathophysiological phenotypes. Finally, we report that, in addition to their structural role, one of the mitoribosomal [2Fe-2S] clusters and the [4Fe-4S] cluster in mitoribosome assembly factor METTL17 sense changes in the redox environment, thus providing a way to regulate organellar protein synthesis accordingly.
Assuntos
Proteínas Ferro-Enxofre , Doenças Mitocondriais , Ribossomos Mitocondriais , Humanos , Proteínas de Transporte/metabolismo , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Metiltransferases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Enxofre/metabolismo , Doenças Mitocondriais/metabolismoRESUMO
LONP1, an AAA+ mitochondrial protease, is implicated in protein quality control, but its precise role in this process remains poorly understood. In this study, we have investigated the role of human LONP1 in mitochondrial proteostasis and gene expression. Depletion of LONP1 resulted in partial loss of mitochondrial DNA (mtDNA) and a complete suppression of mitochondrial translation associated with impaired ribosome biogenesis. The levels of a distinct subset of mitochondrial matrix proteins (SSBP1, MTERFD3, FASTKD2, and CLPX) increased in the presence of a catalytically dead form of LONP1, suggesting that they are bona fide LONP1 substrates. Unexpectedly, the unprocessed forms of the same proteins also accumulated in an insoluble protein fraction. This subset of unprocessed matrix proteins (but not their mature forms) accumulated following depletion of the mitochondrial processing peptidase MPP, though all other MPP substrates investigated were processed normally. Prolonged depletion of LONP1 produced massive matrix protein aggregates, robustly activated the integrated stress response (ISR) pathway, and resulted in stabilization of PINK1, a mitophagy marker. These results demonstrate that LONP1 and MPPαß are together required for the maturation of a subset of LONP1 client proteins and that LONP1 activity is essential for the maintenance of mitochondrial proteostasis and gene expression.
Assuntos
Proteases Dependentes de ATP/metabolismo , Proteínas Mitocondriais/metabolismo , Proteases Dependentes de ATP/antagonistas & inibidores , Proteases Dependentes de ATP/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Agregados Proteicos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteostase , RNA Interferente Pequeno/genética , Estresse Fisiológico , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Peptidase de Processamento MitocondrialRESUMO
The French-Canadian variant of Leigh Syndrome (LSFC) is an autosomal recessive oxidative phosphorylation (OXPHOS) disorder caused by a mutation in LRPPRC, coding for a protein involved in the stability of mitochondrially-encoded mRNAs. Low levels of LRPPRC are present in all patient tissues, but result in a disproportionately severe OXPHOS defect in the brain and liver, leading to unpredictable subacute metabolic crises. To investigate the impact of the OXPHOS defect in the liver, we analyzed the mitochondrial phenotype in mice harboring an hepatocyte-specific inactivation of Lrpprc. Loss of LRPPRC in the liver caused a generalized growth delay, and typical histological features of mitochondrial hepatopathy. At the molecular level, LRPPRC deficiency caused destabilization of polyadenylated mitochondrial mRNAs, altered mitochondrial ultrastructure, and a severe complex IV (CIV) and ATP synthase (CV) assembly defect. The impact of LRPPRC deficiency was not limited to OXPHOS, but also included impairment of long-chain fatty acid oxidation, a striking dysregulation of the mitochondrial permeability transition pore, and an unsuspected alteration of trans-membrane H2O2 diffusion, which was traced to the ATP synthase assembly defect, and to changes in the lipid composition of mitochondrial membranes. This study underscores the value of mitochondria phenotyping to uncover complex and unexpected mechanisms contributing to the pathophysiology of mitochondrial disorders.
Assuntos
Mitocôndrias/metabolismo , Proteínas de Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Permeabilidade da Membrana Celular/genética , Deficiência de Citocromo-c Oxidase/genética , Deficiência de Citocromo-c Oxidase/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Feminino , Hepatócitos/metabolismo , Doença de Leigh/genética , Doença de Leigh/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Fosforilação Oxidativa , Poliadenilação , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA MitocondrialRESUMO
Vitamin B12 (cobalamin, Cbl) cofactors adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) are required for the activity of the enzymes methylmalonyl-CoA mutase (MCM) and methionine synthase (MS). Inborn errors of Cbl metabolism are rare Mendelian disorders associated with hematological and neurological manifestations, and elevations of methylmalonic acid and/or homocysteine in the blood and urine. We describe a patient whose fibroblasts had decreased functional activity of MCM and MS and decreased synthesis of AdoCbl and MeCbl (3.4% and 1.0% of cellular Cbl, respectively). The defect in cultured patient fibroblasts complemented those from all known complementation groups. Patient cells accumulated transcobalamin-bound-Cbl, a complex which usually dissociates in the lysosome to release free Cbl. Whole-exome sequencing identified putative disease-causing variants c.851T>G (p.L284*) and c.1019C>T (p.T340I) in transcription factor ZNF143. Proximity biotinylation analysis confirmed the interaction between ZNF143 and HCFC1, a protein that regulates expression of the Cbl trafficking enzyme MMACHC. qRT-PCR analysis revealed low MMACHC expression levels both in patient fibroblasts, and in control fibroblasts incubated with ZNF143 siRNA.
Assuntos
Citoplasma/metabolismo , Erros Inatos do Metabolismo/genética , Transativadores/genética , Transcobalaminas/metabolismo , Vitamina B 12/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Lactente , Masculino , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/patologia , Mutação , Oxirredutases , LinhagemRESUMO
Protein synthesis in mitochondria is initiated by formylmethionyl-tRNA(Met) (fMet-tRNA(Met)), which requires the activity of the enzyme MTFMT to formylate the methionyl group. We investigated the molecular consequences of mutations in MTFMT in patients with Leigh syndrome or cardiomyopathy. All patients studied were compound heterozygotes. Levels of MTFMT in patient fibroblasts were almost undetectable by immunoblot analysis, and BN-PAGE analysis showed a combined oxidative phosphorylation (OXPHOS) assembly defect involving complexes I, IV and V. The synthesis of only a subset of mitochondrial polypeptides (ND5, ND4, ND1, COXII) was decreased, whereas all others were translated at normal or even increased rates. Expression of the wild-type cDNA rescued the biochemical phenotype when MTFMT was expressed near control levels, but overexpression produced a dominant-negative phenotype, completely abrogating assembly of the OXPHOS complexes, suggesting that MTFMT activity must be tightly regulated. fMet-tRNA(Met) was almost undetectable in control cells and absent in patient cells by high-resolution northern blot analysis, but accumulated in cells overexpressing MTFMT. Newly synthesized COXI was under-represented in complex IV immunoprecipitates from patient fibroblasts, and two-dimensional BN-PAGE analysis of newly synthesized mitochondrial translation products showed an accumulation of free COXI. Quantitative mass spectrophotometry of an N-terminal COXI peptide showed that the ratio of formylated to unmodified N-termini in the assembled complex IV was â¼350:1 in controls and 4:1 in patient cells. These results show that mitochondrial protein synthesis can occur with inefficient formylation of methionyl-tRNA(Met), but that assembly of complex IV is impaired if the COXI N-terminus is not formylated.
Assuntos
Ciclo-Oxigenase 1/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metionina/química , Células Cultivadas , Cromatografia Líquida , Ciclo-Oxigenase 1/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Exoma , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Heterozigoto , Humanos , Doença de Leigh/genética , Mitocôndrias/metabolismo , Mutação , Fosforilação Oxidativa , Biossíntese de Proteínas , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Análise de Sequência de DNA , Espectrometria de Massas em TandemRESUMO
Addition of the trinucleotide cytosine/cytosine/adenine (CCA) to the 3' end of transfer RNAs (tRNAs) is essential for translation and is catalyzed by the enzyme TRNT1 (tRNA nucleotidyl transferase), which functions in both the cytoplasm and mitochondria. Exome sequencing revealed TRNT1 mutations in two unrelated subjects with different clinical features. The first presented with acute lactic acidosis at 3 weeks of age and developed severe developmental delay, hypotonia, microcephaly, seizures, progressive cortical atrophy, neurosensorial deafness, sideroblastic anemia and renal Fanconi syndrome, dying at 21 months. The second presented at 3.5 years with gait ataxia, dysarthria, gross motor regression, hypotonia, ptosis and ophthalmoplegia and had abnormal signals in brainstem and dentate nucleus. In subject 1, muscle biopsy showed combined oxidative phosphorylation (OXPHOS) defects, but there was no OXPHOS deficiency in fibroblasts from either subject, despite a 10-fold-reduction in TRNT1 protein levels in fibroblasts of the first subject. Furthermore, in normal controls, TRNT1 protein levels are 10-fold lower in muscle than in fibroblasts. High resolution northern blots of subject fibroblast RNA suggested incomplete CCA addition to the non-canonical mitochondrial tRNA(Ser(AGY)), but no obvious qualitative differences in other mitochondrial or cytoplasmic tRNAs. Complete knockdown of TRNT1 in patient fibroblasts rendered mitochondrial tRNA(Ser(AGY)) undetectable, and markedly reduced mitochondrial translation, except polypeptides lacking Ser(AGY) codons. These data suggest that the clinical phenotypes associated with TRNT1 mutations are largely due to impaired mitochondrial translation, resulting from defective CCA addition to mitochondrial tRNA(Ser(AGY)), and that the severity of this biochemical phenotype determines the severity and tissue distribution of clinical features.
Assuntos
Mitocôndrias/genética , Doenças Mitocondriais/genética , Mutação , Biossíntese de Proteínas/genética , RNA Nucleotidiltransferases/genética , RNA de Transferência de Serina/metabolismo , Criança , Pré-Escolar , Exoma , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Mitocôndrias/metabolismo , RNA Nucleotidiltransferases/metabolismo , Análise de Sequência de DNA , SíndromeRESUMO
RMND1 is an integral inner membrane mitochondrial protein that assembles into a large 240 kDa complex to support translation of the 13 polypeptides encoded on mtDNA, all of which are essential subunits of the oxidative phosphorylation (OXPHOS) complexes. Variants in RMND1 produce global defects in mitochondrial translation and were first reported in patients with severe neurological phenotypes leading to mortality in the first months of life. Using whole-exome sequencing, we identified compound heterozygous RMND1 variants in a 4-year-old patient with congenital lactic acidosis, severe myopathy, hearing loss, renal failure, and dysautonomia. The levels of mitochondrial ribosome proteins were reduced in patient fibroblasts, causing a translation defect, which was rescued by expression of the wild-type cDNA. RMND1 was almost undetectable by immunoblot analysis in patient muscle and fibroblasts. BN-PAGE analysis showed a severe combined OXPHOS assembly defect that was more prominent in patient muscle than in fibroblasts. Immunofluorescence experiments showed that RMND1 localizes to discrete foci in the mitochondrial network, juxtaposed to RNA granules where the primary mitochondrial transcripts are processed. RMND1 foci were not detected in patient fibroblasts. We hypothesize that RMND1 acts to anchor or stabilize the mitochondrial ribosome near the sites where the mRNAs are matured, spatially coupling post-transcriptional handling mRNAs with their translation, and that loss of function variants in RMND1 are associated with a unique constellation of clinical phenotypes that vary with the severity of the mitochondrial translation defect.
Assuntos
Acidose Láctica/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Surdez/genética , Predisposição Genética para Doença/genética , Insuficiência de Múltiplos Órgãos/genética , Insuficiência Renal/genética , Pré-Escolar , Variação Genética/genética , Humanos , MasculinoRESUMO
French Canadian Leigh Syndrome (LSFC) is an early-onset, progressive neurodegenerative disorder with a distinct pattern of tissue involvement. Most cases are caused by a founder missense mutation in LRPPRC. LRPPRC forms a ribonucleoprotein complex with SLIRP, another RNA-binding protein, and this stabilizes polyadenylated mitochondrial mRNAs. LSFC fibroblasts have reduced levels of LRPPRC and a specific complex IV assembly defect; however, further depletion of mutant LRPPRC results in a complete failure to assemble a functional oxidative phosphorylation system, suggesting that LRPPRC levels determine the nature of the biochemical phenotype. We tested this hypothesis in cultured muscle cells and tissues from LSFC patients. LRPPRC levels were reduced in LSFC muscle cells, resulting in combined complex I and IV deficiencies. A similar combined deficiency was observed in skeletal muscle. Complex IV was only moderately reduced in LSFC heart, but was almost undetectable in liver. Both of these tissues showed elevated levels of complexes I and III. Despite the marked biochemical differences, the steady-state levels of LRPPRC and mitochondrial mRNAs were extremely low, LRPPRC was largely detergent-insoluble, and SLIRP was undetectable in all LSFC tissues. The level of the LRPPRC/SLIRP complex appeared much reduced in control tissues by the first dimension blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis compared with fibroblasts, and even by second dimension analysis it was virtually undetectable in control heart. These results point to tissue-specific pathways for the post-transcriptional handling of mitochondrial mRNAs and suggest that the biochemical defects in LSFC reflect the differential ability of tissues to adapt to the mutation.
Assuntos
Doença de Leigh/metabolismo , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células Cultivadas , Humanos , Doença de Leigh/genética , Fígado/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Especificidade de Órgãos , Fosforilação Oxidativa , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
Mutations in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short-stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole-exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl-tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations.
Assuntos
Catarata/genética , Nanismo Hipofisário/genética , Perda Auditiva Neurossensorial/genética , Isoleucina-tRNA Ligase/genética , Doença de Leigh/genética , Mutação , Doenças do Sistema Nervoso Periférico/genética , Adulto , Sequência de Aminoácidos , Encéfalo/patologia , Catarata/diagnóstico , Consanguinidade , Análise Mutacional de DNA , Nanismo Hipofisário/diagnóstico , Feminino , Genes Recessivos , Perda Auditiva Neurossensorial/diagnóstico , Humanos , Isoleucina-tRNA Ligase/química , Doença de Leigh/diagnóstico , Imageamento por Ressonância Magnética , Masculino , Dados de Sequência Molecular , Linhagem , Doenças do Sistema Nervoso Periférico/diagnóstico , Fenótipo , Alinhamento de Sequência , SíndromeRESUMO
SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Membrana Celular/metabolismo , ATPases Transportadoras de Cobre , Fibroblastos , Humanos , Transporte de Íons , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Chaperonas Moleculares , Oxirredução , Interferência de RNA , RNA Interferente Pequeno , Transdução de SinaisRESUMO
Mammalian mitochondria harbor a dedicated translation apparatus that is required for the synthesis of 13 mitochondrial DNA (mtDNA)-encoded polypeptides, all of which are essential components of the oxidative phosphorylation (OXPHOS) complexes. Little is known about the mechanism of assembly of the mitoribosomes that catalyze this process. Here we show that C7orf30, a member of the large family of DUF143 proteins, associates with the mitochondrial large ribosomal subunit (mt-LSU). Knockdown of C7orf30 by short hairpin RNA (shRNA) does not alter the sedimentation profile of the mt-LSU, but results in the depletion of several mt-LSU proteins and decreased monosome formation. This leads to a mitochondrial translation defect, involving the majority of mitochondrial polypeptides, and a severe OXPHOS assembly defect. Immunoprecipitation and mass spectrometry analyses identified mitochondrial ribosomal protein (MRP)L14 as the specific interacting protein partner of C7orf30 in the mt-LSU. Reciprocal experiments in which MRPL14 was depleted by small interfering RNA (siRNA) phenocopied the C7orf30 knockdown. Members of the DUF143 family have been suggested to be universally conserved ribosomal silencing factors, acting by sterically inhibiting the association of the small and large ribosomal subunits. Our results demonstrate that, although the interaction between C7orf30 and MRPL14 has been evolutionarily conserved, human C7orf30 is, on the contrary, essential for mitochondrial ribosome biogenesis and mitochondrial translation.
Assuntos
Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Ligação Proteica , RNA Interferente Pequeno/genética , Proteínas Ribossômicas/genéticaRESUMO
Complex I (CI, NADH ubiquinone oxidoreductase), the largest complex of the respiratory chain, is composed of 45 structural subunits, 7 of which are encoded in mtDNA. At least 10 factors necessary for holoenzyme assembly have been identified; however, the specific roles of most of them are not well understood. We investigated the role of NDUFAF3, NDUFAF4, C8orf38 and C20orf7, four early assembly factors, in the translation of the mtDNA-encoded CI structural subunits. Transient, or stable, siRNA-mediated knock-down of any of these factors abrogated the assembly of CI, and resulted in a specific decrease in the labeling of the ND1 subunit in a pulse translation experiment, whereas knock-down of NDUFAF2, a late assembly factor, did not affect ND1 translation. Pulse-chase experiments in cells knocked down for NDUFAF3 showed that the half-life of ND1 in the chase was reduced 4-fold, fully accounting for the decrease in pulse labeling. Transient, short-term knock-down of the m-AAA protease AGF3L2 in cells that had been depleted of any of the early CI assembly factors completely rescued the ND1 labeling phenotype, confirming that it is not a synthesis defect, but rather results from rapid proteolysis of newly synthesized ND1. NDUFAF3 co-immunoprecipitated with NDUFAF4, and three matrix arm structural subunits (NDUFS2, NDUFA9, NDUFS3) that are found in a 400 kDa assembly intermediate containing ND1. These data suggest that the four early CI assembly factors have non-redundant functions in the assembly of a module that docks and stabilizes newly synthesized ND1, nucleating assembly of the holoenzyme.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Renovação Mitocondrial , NADH Desidrogenase/metabolismo , Subunidades Proteicas/metabolismo , Linhagem Celular , Eletroforese em Gel Bidimensional , Técnicas de Silenciamento de Genes , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Peptídeos/metabolismo , Fenótipo , Biossíntese de Proteínas , Coloração e RotulagemRESUMO
The mammalian mitochondrial genome contains 37 genes, 13 of which encode polypeptide subunits in the enzyme complexes of the oxidative phosphorylation system. The other genes encode the rRNAs and tRNAs necessary for their translation. The mitochondrial translation machinery is located in the mitochondrial matrix, and is exclusively dedicated to the synthesis of these 13 enzyme subunits. Mitochondrial disease in humans is often associated with defects in mitochondrial translation. This can manifest as a global decrease in the rate of mitochondrial protein synthesis, a decrease in the synthesis of specific polypeptides, the synthesis of abnormal polypeptides, or in altered stability of specific translation products. All of these changes in the normal pattern of mitochondrial translation can be assessed by a straightforward technique that takes advantage of the insensitivity of the mitochondrial translation machinery to antibiotics that completely inhibit cytoplasmic translation. Thus, specific radioactive labeling of the mitochondrial translation products can be achieved in cultured cells, and the results can be visualized on gradient gels. The analysis of mitochondrial translation in cells cultured from patient biopsies is useful in the study of disease-causing mutations in both the mitochondrial and the nuclear genomes.
Assuntos
Marcação por Isótopo/métodos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Genoma Mitocondrial/genética , Humanos , Metionina/metabolismo , Proteínas Mitocondriais/isolamento & purificação , Mioblastos/citologia , Mioblastos/metabolismo , Radioisótopos de EnxofreRESUMO
MTU1 (TRMU) is a mitochondrial enzyme responsible for the 2-thiolation of the wobble U in tRNA(Lys), tRNA(Glu) and tRNA(Gln), a post-transcriptional modification believed to be important for accurate and efficient synthesis of the 13 respiratory chain subunits encoded by mtDNA. Mutations in MTU1 are associated with acute infantile liver failure, and this has been ascribed to a transient lack of cysteine, the sulfur donor for the thiouridylation reaction, resulting in a mitochondrial translation defect during early development. A mutation in tRNA(Lys) that causes myoclonic epilepsy with ragged-red fibers (MERRF) is also reported to prevent modification of the wobble U. Here we show that mitochondrial translation is unaffected in fibroblasts from an MTU1 patient, in which MTU1 is undetectable by immunoblotting, despite the severe reduction in the 2-thiolation of mitochondrial tRNA(Lys), tRNA(Glu) and tRNA(Gln). The only respiratory chain abnormality that we could observe in these cells was an accumulation of a Complex II assembly intermediate, which, however, did not affect the level of the fully assembled enzyme. The identical phenotype was observed by siRNA-mediated knockdown of MTU1 in HEK 293 cells. Further, the mitochondrial translation deficiencies present in myoblasts from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episode and MERRF patients, which are associated with defects in post-transcriptional modification of mitochondrial tRNAs, did not worsen following knockdown of MTU1 in these cells. This study demonstrates that MTU1 is not required for mitochondrial translation at normal steady-state levels of tRNAs, and that it may possess an as yet uncharacterized function in another sulfur-trafficking pathway.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas , tRNA Metiltransferases/metabolismo , Eletroforese em Gel de Poliacrilamida , Fibroblastos/enzimologia , Fibroblastos/patologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Síndrome MELAS/enzimologia , Síndrome MELAS/patologia , Síndrome MERRF/enzimologia , Síndrome MERRF/patologia , Proteínas Mitocondriais/deficiência , Mutação/genética , Mioblastos/enzimologia , Mioblastos/patologia , Fosforilação Oxidativa , RNA de Transferência/metabolismo , Tiouridina/metabolismo , tRNA Metiltransferases/deficiênciaRESUMO
Severe combined deficiency of the 2-oxoacid dehydrogenases, associated with a defect in lipoate synthesis and accompanied by defects in complexes I, II, and III of the mitochondrial respiratory chain, is a rare autosomal recessive syndrome with no obvious causative gene defect. A candidate locus for this syndrome was mapped to chromosomal region 2p14 by microcell-mediated chromosome transfer in two unrelated families. Unexpectedly, analysis of genes in this area identified mutations in two different genes, both of which are involved in [Fe-S] cluster biogenesis. A homozygous missense mutation, c.545G>A, near the splice donor of exon 6 in NFU1 predicting a p.Arg182Gln substitution was found in one of the families. The mutation results in abnormal mRNA splicing of exon 6, and no mature protein could be detected in fibroblast mitochondria. A single base-pair duplication c.123dupA was identified in BOLA3 in the second family, causing a frame shift that produces a premature stop codon (p.Glu42Argfs(∗)13). Transduction of fibroblast lines with retroviral vectors expressing the mitochondrial, but not the cytosolic isoform of NFU1 and with isoform 1, but not isoform 2 of BOLA3 restored both respiratory chain function and oxoacid dehydrogenase complexes. NFU1 was previously proposed to be an alternative scaffold to ISCU for the biogenesis of [Fe-S] centers in mitochondria, and the function of BOLA3 was previously unknown. Our results demonstrate that both play essential roles in the production of [Fe-S] centers for the normal maturation of lipoate-containing 2-oxoacid dehydrogenases, and for the assembly of the respiratory chain complexes.
Assuntos
Proteínas de Transporte/genética , Mutação , Oxirredutases/metabolismo , Proteínas/genética , Citosol/metabolismo , Transporte de Elétrons , Éxons , Saúde da Família , Feminino , Fibroblastos/metabolismo , Homozigoto , Humanos , Proteínas Ferro-Enxofre/metabolismo , Masculino , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Mutação de Sentido IncorretoRESUMO
Mitochondrial DNA (mtDNA) sequence variants segregate in mutation and tissue-specific manners, but the mechanisms remain unknown. The segregation pattern of pathogenic mtDNA mutations is a major determinant of the onset and severity of disease. Using a heteroplasmic mouse model, we demonstrate that Gimap3, an outer mitochondrial membrane GTPase, is a critical regulator of this process in leukocytes. Gimap3 is important for T cell development and survival, suggesting that leukocyte survival may be a key factor in the genetic regulation of mtDNA sequence variants and in modulating human mitochondrial diseases.
Assuntos
DNA Mitocondrial/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Haplótipos/genética , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Mamíferos/citologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Sistema Hematopoético/metabolismo , Humanos , Rim/metabolismo , Leucócitos/citologia , Leucócitos/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Baço/metabolismoRESUMO
Mutations in LRPPRC are responsible for the French Canadian variant of Leigh syndrome (LSFC), a neurodegenerative disorder caused by a tissue-specific deficiency in cytochrome c oxidase (COX). To investigate the pathogenic mechanism of disease, we studied LRPPRC function in LSFC and control fibroblasts. The level of mutated LRPPRC is reduced in LSFC cells, and this results in decreased steady-state levels of most mitochondrial mRNAs, but not rRNAs or tRNAs, a phenotype that can be reproduced by siRNA-mediated knockdown of LRPPRC in control cells. Processing of the primary transcripts appears normal. The resultant defect in mitochondrial protein synthesis in LSFC cells disproportionately affects the COX subunits, leading to an isolated COX assembly defect. Further knockdown of LRPPRC produces a generalized assembly defect in all oxidative phosphorylation complexes containing mtDNA-encoded subunits, due to a severe decrease in all mitochondrial mRNAs. LRPPRC exists in a high-molecular-weight complex, and it coimmunoprecipitates with SLIRP, a stem-loop RNA-binding protein. Although this interaction does not depend on mitochondrial mRNA, both proteins show reduced stability in its absence. These results implicate LRPPRC in posttranscriptional mitochondrial gene expression as part of a ribonucleoprotein complex that regulates the stability and handling of mature mRNAs.
Assuntos
Regulação da Expressão Gênica , Mitocôndrias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Cinética , Doença de Leigh/enzimologia , Doença de Leigh/genética , Doença de Leigh/patologia , Mitocôndrias/patologia , Peso Molecular , Complexos Multiproteicos/metabolismo , Proteínas Mutantes/metabolismo , Fosforilação Oxidativa , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismoRESUMO
Human SCO1 and SCO2 code for essential metallochaperones with ill-defined functions in the biogenesis of the CuA site of cytochrome c oxidase subunit II (CO II). Here, we have used patient cell lines to investigate the specific roles of each SCO protein in this pathway. By pulse-labeling mitochondrial translation products, we demonstrate that the synthesis of CO II is reduced in SCO2, but not in SCO1, cells. Despite this biosynthetic defect, newly synthesized CO II is more stable in SCO2 cells than in control cells. RNAi-mediated knockdown of mutant SCO2 abolishes CO II labeling in the translation assay, whereas knockdown of mutant SCO1 does not affect CO II synthesis. These results indicate that SCO2 acts upstream of SCO1, and that it is indispensable for CO II synthesis. The subsequent maturation of CO II is contingent upon the formation of a complex that includes both SCO proteins, each with a functional CxxxC copper-coordinating motif. In control cells, the cysteines in this motif in SCO1 exist as a mixed population comprised of oxidized disulphides and reduced thiols; however, the relative ratio of oxidized to reduced cysteines in SCO1 is perturbed in cells from both SCO backgrounds. Overexpression of wild-type SCO2, or knockdown of mutant SCO2, in SCO2 cells alters the ratio of oxidized to reduced cysteines in SCO1, suggesting that SCO2 acts as a thiol-disulphide oxidoreductase to oxidize the copper-coordinating cysteines in SCO1 during CO II maturation. Based on these data we present a model in which each SCO protein fulfills distinct, stage-specific functions during CO II synthesis and CuA site maturation.