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1.
EMBO Rep ; 21(1): e48833, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31721420

RESUMO

The mitochondrial genome encodes for thirteen core subunits of the oxidative phosphorylation system. These proteins assemble with imported proteins in a modular manner into stoichiometric enzyme complexes. Assembly factors assist in these biogenesis processes by providing co-factors or stabilizing transient assembly stages. However, how expression of the mitochondrial-encoded subunits is regulated to match the availability of nuclear-encoded subunits is still unresolved. Here, we address the function of MITRAC15/COA1, a protein that participates in complex I biogenesis and complex IV biogenesis. Our analyses of a MITRAC15 knockout mutant reveal that MITRAC15 is required for translation of the mitochondrial-encoded complex I subunit ND2. We find that MITRAC15 is a constituent of a ribosome-nascent chain complex during ND2 translation. Chemical crosslinking analyses demonstrate that binding of the ND2-specific assembly factor ACAD9 to the ND2 polypeptide occurs at the C-terminus and thus downstream of MITRAC15. Our analyses demonstrate that expression of the founder subunit ND2 of complex I undergoes regulation. Moreover, a ribosome-nascent chain complex with MITRAC15 is at the heart of this process.

2.
Nucleic Acids Res ; 46(16): 8471-8482, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30085210

RESUMO

The human mitochondrial translation apparatus, which synthesizes the core subunits of the oxidative phosphorylation system, is of central interest as mutations in several genes encoding for mitoribosomal proteins or translation factors cause severe human diseases. Little is known, how this complex machinery assembles from nuclear-encoded protein components and mitochondrial-encoded RNAs, and which ancillary factors are required to form a functional mitoribosome. We have characterized the human Obg protein GTPBP10, which associates specifically with the mitoribosomal large subunit at a late maturation state. Defining its interactome, we have shown that GTPBP10 is in a complex with other mtLSU biogenesis factors including mitochondrial RNA granule components, the 16S rRNA module and late mtLSU assembly factors such as MALSU1, SMCR7L, MTERF4 and NSUN4. GTPBP10 deficiency leads to a drastic reduction in 55S monosome formation resulting in defective mtDNA-expression and in a decrease in cell growth. Our results suggest that GTPBP10 is a ribosome biogenesis factor of the mtLSU required for late stages of maturation.


Assuntos
Ribossomos Mitocondriais/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , DNA Mitocondrial/genética , Humanos , Metiltransferases/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Fosforilação Oxidativa , RNA Ribossômico 16S/genética , Fatores de Transcrição/genética
3.
Elife ; 72018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29381136

RESUMO

Cytochrome c oxidase of the mitochondrial oxidative phosphorylation system reduces molecular oxygen with redox equivalent-derived electrons. The conserved mitochondrial-encoded COX1- and COX2-subunits are the heme- and copper-center containing core subunits that catalyze water formation. COX1 and COX2 initially follow independent biogenesis pathways creating assembly modules with subunit-specific, chaperone-like assembly factors that assist in redox centers formation. Here, we find that COX16, a protein required for cytochrome c oxidase assembly, interacts specifically with newly synthesized COX2 and its copper center-forming metallochaperones SCO1, SCO2, and COA6. The recruitment of SCO1 to the COX2-module is COX16- dependent and patient-mimicking mutations in SCO1 affect interaction with COX16. These findings implicate COX16 in CuA-site formation. Surprisingly, COX16 is also found in COX1-containing assembly intermediates and COX2 recruitment to COX1. We conclude that COX16 participates in merging the COX1 and COX2 assembly lines.


Assuntos
Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Transporte/metabolismo , Ciclo-Oxigenase 1/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Multimerização Proteica
4.
Biochem J ; 474(13): 2145-2158, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28512204

RESUMO

Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis. Unlike its bacterial orthologue, RBFA associates mainly with helices 44 and 45 of the 12S rRNA in the mitoribosomal small subunit to promote dimethylation of two highly conserved consecutive adenines. Characterization of RBFA-depleted cells indicates that this dimethylation is not a prerequisite for assembly of the small ribosomal subunit. However, the RBFA-facilitated modification is necessary for completing mt-rRNA maturation and regulating association of the small and large subunits to form a functional monosome implicating RBFA in the quality control of mitoribosome formation.


Assuntos
Proteínas de Escherichia coli/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Proteínas de Escherichia coli/genética , Células HEK293 , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , RNA Ribossômico/genética , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Homologia de Sequência de Aminoácidos
5.
Cell ; 167(2): 471-483.e10, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27693358

RESUMO

Mitochondrial ribosomes translate membrane integral core subunits of the oxidative phosphorylation system encoded by mtDNA. These translation products associate with nuclear-encoded, imported proteins to form enzyme complexes that produce ATP. Here, we show that human mitochondrial ribosomes display translational plasticity to cope with the supply of imported nuclear-encoded subunits. Ribosomes expressing mitochondrial-encoded COX1 mRNA selectively engage with cytochrome c oxidase assembly factors in the inner membrane. Assembly defects of the cytochrome c oxidase arrest mitochondrial translation in a ribosome nascent chain complex with a partially membrane-inserted COX1 translation product. This complex represents a primed state of the translation product that can be retrieved for assembly. These findings establish a mammalian translational plasticity pathway in mitochondria that enables adaptation of mitochondrial protein synthesis to the influx of nuclear-encoded subunits.


Assuntos
Ciclo-Oxigenase 1/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 1/genética , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Fosforilação Oxidativa , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mitocondrial , Ribossomos/metabolismo
6.
EMBO Rep ; 17(12): 1844-1856, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27737933

RESUMO

The SPFH (stomatin, prohibitin, flotillin, HflC/K) superfamily is composed of scaffold proteins that form ring-like structures and locally specify the protein-lipid composition in a variety of cellular membranes. Stomatin-like protein 2 (SLP2) is a member of this superfamily that localizes to the mitochondrial inner membrane (IM) where it acts as a membrane organizer. Here, we report that SLP2 anchors a large protease complex composed of the rhomboid protease PARL and the i-AAA protease YME1L, which we term the SPY complex (for SLP2-PARL-YME1L). Association with SLP2 in the SPY complex regulates PARL-mediated processing of PTEN-induced kinase PINK1 and the phosphatase PGAM5 in mitochondria. Moreover, SLP2 inhibits the stress-activated peptidase OMA1, which can bind to SLP2 and cleaves PGAM5 in depolarized mitochondria. SLP2 restricts OMA1-mediated processing of the dynamin-like GTPase OPA1 allowing stress-induced mitochondrial hyperfusion under starvation conditions. Together, our results reveal an important role of SLP2 membrane scaffolds for the spatial organization of IM proteases regulating mitochondrial dynamics, quality control, and cell survival.


Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Metaloproteases/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Proteínas Sanguíneas/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Metaloproteases/genética , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Peptídeo Hidrolases/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteólise
7.
Mol Cell ; 64(1): 148-162, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27642048

RESUMO

Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca2+ homeostasis.


Assuntos
Canais de Cálcio/metabolismo , Cerebelo/metabolismo , Corpo Estriado/metabolismo , Hipocampo/metabolismo , Metaloendopeptidases/genética , Mitocôndrias/metabolismo , Neurônios/metabolismo , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Morte Celular , Cerebelo/patologia , Corpo Estriado/patologia , Regulação da Expressão Gênica , Células HEK293 , Hipocampo/patologia , Homeostase/genética , Humanos , Transporte de Íons , Metaloendopeptidases/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neurônios/patologia , Mapeamento de Interação de Proteínas , Transdução de Sinais
8.
J Cell Biol ; 212(2): 157-66, 2016 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-26783299

RESUMO

Proteolytic cleavage of the dynamin-like guanosine triphosphatase OPA1 in mitochondria is emerging as a central regulatory hub that determines mitochondrial morphology under stress and in disease. Stress-induced OPA1 processing by OMA1 triggersmitochondrial fragmentation, which is associated with mitophagy and apoptosis in vitro. Here, we identify OMA1 as a critical regulator of neuronal survival in vivo and demonstrate that stress-induced OPA1 processing by OMA1 promotes neuronal death and neuroinflammatory responses. Using mice lacking prohibitin membrane scaffolds as a model of neurodegeneration, we demonstrate that additional ablation of Oma1 delays neuronal loss and prolongs lifespan. This is accompanied by the accumulation of fusion-active, long OPA1 forms, which stabilize the mitochondrial genome but do not preserve mitochondrial cristae or respiratory chain supercomplex assembly in prohibitin-depleted neurons. Thus, long OPA1 forms can promote neuronal survival independently of cristae shape, whereas stress-induced OMA1 activation and OPA1 cleavage limit mitochondrial fusion and promote neuronal death.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Metaloproteases/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Degeneração Neural , Animais , Apoptose , Encéfalo/metabolismo , Encéfalo/patologia , Respiração Celular , Sobrevivência Celular/genética , Células Cultivadas , DNA Mitocondrial/metabolismo , Deleção de Genes , Metaloproteases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Degeneração Neural/genética , Neurônios/metabolismo , Neurônios/patologia , Proteínas Repressoras/metabolismo
9.
Nat Rev Mol Cell Biol ; 16(10): 586-92, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26535422

RESUMO

Mitochondrial-encoded subunits of the oxidative phosphorylation system assemble with nuclear-encoded subunits into enzymatic complexes. Recent findings showed that mitochondrial translation is linked to other mitochondrial functions, as well as to cellular processes. The supply of mitochondrial-encoded proteins is coordinated by the coupling of mitochondrial protein synthesis with assembly of respiratory chain complexes. MicroRNAs imported from the cytoplasm into mitochondria were, surprisingly, found to act as regulators of mitochondrial translation. In turn, translation in mitochondria controls cellular proliferation, and mitochondrial ribosomal subunits contribute to the cytoplasmic stress response. Thus, translation in mitochondria is apparently integrated into cellular processes.


Assuntos
MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Biossíntese de Proteínas/fisiologia , RNA/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Transporte de Elétrons/fisiologia , Humanos , MicroRNAs/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , RNA/genética , RNA Mitocondrial
10.
Front Microbiol ; 5: 374, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25101074

RESUMO

Protein synthesis is central to life and maintaining a highly accurate and efficient mechanism is essential. What happens when a translating ribosome stalls on a messenger RNA? Many highly intricate processes have been documented in the cytosol of numerous species, but how does organellar protein synthesis resolve this stalling issue? Mammalian mitochondria synthesize just thirteen highly hydrophobic polypeptides. These proteins are all integral components of the machinery that couples oxidative phosphorylation. Consequently, it is essential that stalled mitochondrial ribosomes can be efficiently recycled. To date, there is no evidence to support any particular molecular mechanism to resolve this problem. However, here we discuss the observation that there are four predicted members of the mitochondrial translation release factor family and that only one member, mtRF1a, is necessary to terminate the translation of all thirteen open reading frames in the mitochondrion. Could the other members be involved in the process of recycling stalled mitochondrial ribosomes?

11.
Cell Metab ; 20(1): 158-71, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24856930

RESUMO

Prohibitins form large protein and lipid scaffolds in the inner membrane of mitochondria that are required for mitochondrial morphogenesis, neuronal survival, and normal lifespan. Here, we have defined the interactome of PHB2 in mitochondria and identified DNAJC19, mutated in dilated cardiomyopathy with ataxia, as binding partner of PHB complexes. We observed impaired cell growth, defective cristae morphogenesis, and similar transcriptional responses in the absence of either DNAJC19 or PHB2. The loss of PHB/DNAJC19 complexes affects cardiolipin acylation and leads to the accumulation of cardiolipin species with altered acyl chains. Similar defects occur in cells lacking the transacylase tafazzin, which is mutated in Barth syndrome. Our experiments suggest that PHB/DNAJC19 membrane domains regulate cardiolipin remodeling by tafazzin and explain similar clinical symptoms in two inherited cardiomyopathies by an impaired cardiolipin metabolism in mitochondrial membranes.


Assuntos
Cardiolipinas/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sequência de Aminoácidos , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Linhagem Celular , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Metaloendopeptidases/metabolismo , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/genética , Membranas Mitocondriais/metabolismo , Dados de Sequência Molecular , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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