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1.
Cell Rep ; 10(7): 1110-21, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25704814

RESUMO

The mitochondrial genome relies heavily on post-transcriptional events for its proper expression, and misregulation of this process can cause mitochondrial genetic diseases in humans. Here, we report that a novel translational variant of Fas-activated serine/threonine kinase (FASTK) co-localizes with mitochondrial RNA granules and is required for the biogenesis of ND6 mRNA, a mitochondrial-encoded subunit of the NADH dehydrogenase complex (complex I). We show that ablating FASTK expression in cultured cells and mice results specifically in loss of ND6 mRNA and reduced complex I activity in vivo. FASTK binds at multiple sites along the ND6 mRNA and its precursors and cooperates with the mitochondrial degradosome to ensure regulated ND6 mRNA biogenesis. These data provide insights into the mechanism and control of mitochondrial RNA processing within mitochondrial RNA granules.


Assuntos
Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Complexo I de Transporte de Elétrons/metabolismo , Endorribonucleases/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Microscopia Confocal , Complexos Multienzimáticos/metabolismo , Miocárdio/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , RNA Helicases/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Mitocondrial , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
2.
Cell Metab ; 17(3): 399-410, 2013 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-23473034

RESUMO

Various specialized domains have been described in the cytosol and the nucleus; however, little is known about compartmentalization within the mitochondrial matrix. GRSF1 (G-rich sequence factor 1) is an RNA binding protein that was previously reported to localize in the cytosol. We found that an isoform of GRSF1 accumulates in discrete foci in the mitochondrial matrix. These foci are composed of nascent mitochondrial RNA and also contain RNase P, an enzyme that participates in mitochondrial RNA processing. GRSF1 was found to interact with RNase P and to be required for processing of both classical and tRNA-less RNA precursors. In its absence, cleavage of primary RNA transcripts is abnormal, leading to decreased expression of mitochondrially encoded proteins and mitochondrial dysfunction. Our findings suggest that the foci containing GRSF1 and RNase P correspond to sites where primary RNA transcripts converge to be processed. We have termed these large ribonucleoprotein structures "mitochondrial RNA granules."


Assuntos
Regulação da Expressão Gênica/fisiologia , Mitocôndrias/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Northern Blotting , Bromodesoxiuridina , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Mitocondrial , Ribonuclease P/metabolismo
3.
Mol Biol Cell ; 20(19): 4216-24, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19656850

RESUMO

m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits.


Assuntos
Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteases Dependentes de ATP , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Catálise , Núcleo Celular/enzimologia , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Immunoblotting , Masculino , Metaloendopeptidases/genética , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Transfecção , Peptidase de Processamento Mitocondrial
4.
Mol Biol Cell ; 18(9): 3582-90, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17615298

RESUMO

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Sequência de Aminoácidos , Animais , GTP Fosfo-Hidrolases/química , Células HeLa , Humanos , Isoenzimas/química , Metaloendopeptidases/deficiência , Camundongos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Especificidade por Substrato
5.
Crit Rev Biochem Mol Biol ; 42(3): 221-42, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17562452

RESUMO

Cell survival depends on essential processes in mitochondria. Various proteases within these organelles regulate mitochondrial biogenesis and ensure the complete degradation of excess or damaged proteins. Many of these proteases are highly conserved and ubiquitous in eukaryotic cells. They can be assigned to three functional classes: processing peptidases, which cleave off mitochondrial targeting sequences of nuclearly encoded proteins and process mitochondrial proteins with regulatory functions; ATP-dependent proteases, which either act as processing peptidases with regulatory functions or as quality-control enzymes degrading non-native polypeptides to peptides; and oligopeptidases, which degrade these peptides and mitochondrial targeting sequences to amino acids. Disturbances of protein degradation within mitochondria cause severe phenotypes in various organisms and can lead to the induction of apoptotic programmes and cell-specific neurodegeneration in mammals. After an overview of the proteolytic system of mitochondria, we will focus on versatile functions of ATP-dependent AAA proteases in the inner membrane. These conserved proteolytic machines conduct protein quality surveillance of mitochondrial inner membrane proteins, mediate vectorial protein dislocation from membranes, and, acting as processing enzymes, control ribosome assembly, mitochondrial protein synthesis, and mitochondrial fusion. Implications of these functions for cell-specific axonal degeneration in hereditary spastic paraplegia will be discussed.


Assuntos
Metaloendopeptidases/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Humanos , Modelos Biológicos , Degeneração Neural/etiologia , Degeneração Neural/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Paraplegia Espástica Hereditária/etiologia , Paraplegia Espástica Hereditária/metabolismo
6.
Mol Cell Biol ; 27(2): 758-67, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17101804

RESUMO

The m-AAA protease, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, controls protein quality and regulates ribosome assembly, thus exerting essential housekeeping functions within mitochondria. Mutations in the m-AAA protease subunit paraplegin cause axonal degeneration in hereditary spastic paraplegia (HSP), but the basis for the unexpected tissue specificity is not understood. Paraplegin assembles with homologous Afg3l2 subunits into hetero-oligomeric complexes which can substitute for yeast m-AAA proteases, demonstrating functional conservation. The function of a third paralogue, Afg3l1 expressed in mouse, is unknown. Here, we analyze the assembly of paraplegin into m-AAA complexes and monitor consequences of paraplegin deficiency in HSP fibroblasts and in a mouse model for HSP. Our findings reveal variability in the assembly of m-AAA proteases in mitochondria in different tissues. Homo-oligomeric Afg3l1 and Afg3l2 complexes and hetero-oligomeric assemblies of both proteins with paraplegin can be formed. Yeast complementation studies demonstrate the proteolytic activity of these assemblies. Paraplegin deficiency in HSP does not result in the loss of m-AAA protease activity in brain mitochondria. Rather, homo-oligomeric Afg3l2 complexes accumulate, and these complexes can substitute for housekeeping functions of paraplegin-containing m-AAA complexes. We therefore propose that the formation of m-AAA proteases with altered substrate specificities leads to axonal degeneration in HSP.


Assuntos
Metaloendopeptidases/metabolismo , Proteínas Mitocondriais/metabolismo , Paraplegia Espástica Hereditária/metabolismo , Proteases Dependentes de ATP , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/metabolismo , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Humanos , Hidrólise , Metaloendopeptidases/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Paraplegia Espástica Hereditária/genética
7.
Cell ; 123(2): 277-89, 2005 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-16239145

RESUMO

AAA proteases comprise a conserved family of membrane bound ATP-dependent proteases that ensures the quality control of mitochondrial inner-membrane proteins. Inactivation of AAA proteases causes pleiotropic phenotypes in various organisms, including respiratory deficiencies, mitochondrial morphology defects, and axonal degeneration in hereditary spastic paraplegia (HSP). The molecular basis of these defects, however, remained unclear. Here, we describe a regulatory role of an AAA protease for mitochondrial protein synthesis in yeast. The mitochondrial ribosomal protein MrpL32 is processed by the m-AAA protease, allowing its association with preassembled ribosomal particles and completion of ribosome assembly in close proximity to the inner membrane. Maturation of MrpL32 and mitochondrial protein synthesis are also impaired in a HSP mouse model lacking the m-AAA protease subunit paraplegin, demonstrating functional conservation. Our findings therefore rationalize mitochondrial defects associated with m-AAA protease mutants in yeast and shed new light on the mechanism of axonal degeneration in HSP.


Assuntos
Adenosina Trifosfatases/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Paraplegia Espástica Hereditária/enzimologia , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Metaloendopeptidases/química , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Membranas Mitocondriais/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Paraplegia Espástica Hereditária/genética , Especificidade por Substrato
8.
J Cell Biol ; 163(4): 777-87, 2003 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-14623864

RESUMO

Mmutations in paraplegin, a putative mitochondrial metallopeptidase of the AAA family, cause an autosomal recessive form of hereditary spastic paraplegia (HSP). Here, we analyze the function of paraplegin at the cellular level and characterize the phenotypic defects of HSP patients' cells lacking this protein. We demonstrate that paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane. These two proteins form a high molecular mass complex, which we show to be aberrant in HSP fibroblasts. The loss of this complex causes a reduced complex I activity in mitochondria and an increased sensitivity to oxidant stress, which can both be rescued by exogenous expression of wild-type paraplegin. Furthermore, complementation studies in yeast demonstrate functional conservation of the human paraplegin-AFG3L2 complex with the yeast m-AAA protease and assign proteolytic activity to this structure. These results shed new light on the molecular pathogenesis of HSP and functionally link AFG3L2 to this neurodegenerative disease.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Metaloendopeptidases/deficiência , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Paraplegia Espástica Hereditária/enzimologia , Proteases Dependentes de ATP , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Respiração Celular/genética , Células Cultivadas , Regulação para Baixo/genética , Complexo I de Transporte de Elétrons/deficiência , Fibroblastos , Humanos , Membranas Intracelulares/metabolismo , Substâncias Macromoleculares , Metaloendopeptidases/genética , Estresse Oxidativo/genética , Filogenia , Espécies Reativas de Oxigênio/farmacologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência do Ácido Nucleico , Paraplegia Espástica Hereditária/genética
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