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1.
Science ; 344(6191): 1510-5, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24970086

RESUMO

Dynamin superfamily molecular motors use guanosine triphosphate (GTP) as a source of energy for membrane-remodeling events. We found that knockdown of nucleoside diphosphate kinases (NDPKs) NM23-H1/H2, which produce GTP through adenosine triphosphate (ATP)-driven conversion of guanosine diphosphate (GDP), inhibited dynamin-mediated endocytosis. NM23-H1/H2 localized at clathrin-coated pits and interacted with the proline-rich domain of dynamin. In vitro, NM23-H1/H2 were recruited to dynamin-induced tubules, stimulated GTP-loading on dynamin, and triggered fission in the presence of ATP and GDP. NM23-H4, a mitochondria-specific NDPK, colocalized with mitochondrial dynamin-like OPA1 involved in mitochondria inner membrane fusion and increased GTP-loading on OPA1. Like OPA1 loss of function, silencing of NM23-H4 but not NM23-H1/H2 resulted in mitochondrial fragmentation, reflecting fusion defects. Thus, NDPKs interact with and provide GTP to dynamins, allowing these motor proteins to work with high thermodynamic efficiency.


Assuntos
Membrana Celular/metabolismo , Dinaminas/metabolismo , Guanosina Trifosfato/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Fusão de Membrana , Mitocôndrias/metabolismo , Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo Difosfato Quinase D/metabolismo
2.
J Cell Biol ; 187(7): 959-66, 2009 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-20038677

RESUMO

The mammalian mitochondrial inner membrane fusion protein OPA1 is controlled by complex patterns of alternative splicing and proteolysis. A subset of OPA1 isoforms is constitutively cleaved by YME1L. Other isoforms are not cleaved by YME1L, but they are cleaved when mitochondria lose membrane potential or adenosine triphosphate. In this study, we show that this inducible cleavage is mediated by a zinc metalloprotease called OMA1. We find that OMA1 small interfering RNA inhibits inducible cleavage, helps retain fusion competence, and slows the onset of apoptosis, showing that OMA1 controls OPA1 cleavage and function. We also find that OMA1 is normally cleaved from 60 to 40 kD by another as of yet unidentified protease. Loss of membrane potential causes 60-kD protein to accumulate, suggesting that OMA1 is attenuated by proteolytic degradation. We conclude that a proteolytic cascade controls OPA1. Inducible cleavage provides a mechanism for quality control because proteolytic inactivation of OPA1 promotes selective removal of defective mitochondrial fragments by preventing their fusion with the mitochondrial network.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Metaloendopeptidases/fisiologia , Proteínas Mitocondriais/fisiologia , Peptídeo Hidrolases/fisiologia , Apoptose , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Células HeLa , Humanos , Potenciais da Membrana , Metaloendopeptidases/análise , Metaloendopeptidases/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Isoformas de Proteínas/metabolismo , Interferência de RNA
3.
J Cell Biol ; 178(5): 757-64, 2007 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-17709430

RESUMO

The dynamin-related protein Opa1 is localized to the mitochondrial intermembrane space, where it facilitates fusion between mitochondria. Apoptosis causes Opa1 release into the cytosol and causes mitochondria to fragment. Loss of mitochondrial membrane potential also causes mitochondrial fragmentation but not Opa1 release into the cytosol. Both conditions induce the proteolytic cleavage of Opa1, suggesting that mitochondrial fragmentation is triggered by Opa1 inactivation. The opposite effect was observed with knockdown of the mitochondrial intermembrane space protease Yme1. Knockdown of Yme1 prevents the constitutive cleavage of a subset of Opa1 splice variants but does not affect carbonyl cyanide m-chlorophenyl hydrazone or apoptosis-induced cleavage. Knockdown of Yme1 also increases mitochondrial connectivity, but this effect is independent of Opa1 because it also occurs in Opa1 knockdown cells. We conclude that Yme1 constitutively regulates a subset of Opa1 isoforms and an unknown mitochondrial morphology protein, whereas the loss of membrane potential induces the further proteolysis of Opa1.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/fisiologia , Fragmentos de Peptídeos/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Carbonil Cianeto m-Clorofenil Hidrazona/metabolismo , GTP Fosfo-Hidrolases/genética , Células HeLa , Humanos , Ionóforos/metabolismo , Potenciais da Membrana/fisiologia , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Metaloproteases/genética , Metaloproteases/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fragmentos de Peptídeos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
4.
Methods Enzymol ; 404: 620-31, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16413305

RESUMO

Opa1, also known as Mgm1 in yeast, is a mitochondrial member of the dynamin family. Unlike other dynamin family members, Opa1 has an N-terminal mitochondrial targeting sequence, suggesting that this protein is imported into mitochondria. Here, we describe biochemical techniques, such as mitochondrial isolation, digitonin extraction, a protease protection assay, and carbonate extraction, that were used to determine that mammalian Opa1 resides in the intermembrane space where it is tightly bound to the inner membrane. In addition, we describe bacterial expression of the Opa1 GTPase domain, methods for purification, and an in vitro assay for GTP hydrolysis.


Assuntos
GTP Fosfo-Hidrolases/análise , Proteínas Mitocondriais/análise , Animais , Encéfalo/ultraestrutura , Bovinos , Digitonina/farmacologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/fisiologia , Guanosina Trifosfato/metabolismo , Humanos , Mitocôndrias Hepáticas/química , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas Mitocondriais/fisiologia , Mutação , Estrutura Terciária de Proteína/genética
5.
J Biol Chem ; 279(18): 18792-8, 2004 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-14970223

RESUMO

Mgm1 is a member of the dynamin family of GTP-binding proteins. Mgm1 was first identified in yeast, where it affects mitochondrial morphology. The human homologue of Mgm1 is called OPA1. Mutations in the OPA1 gene are the prevailing cause of dominant optic atrophy, a hereditary disease in which progressive degeneration of the optic nerve can lead to blindness. Here we investigate the properties of the Mgm1/OPA1 protein in mammalian cells. We find that Mgm1/OPA1 is localized to the mitochondrial intermembrane space, where it is tightly bound to the outer surface of the inner membrane. Overexpression of wild type or mutant forms of the Mgm1/OPA1 protein cause mitochondria to fragment and, in some cases, cluster near the nucleus, whereas the loss of protein caused by small interfering RNA (siRNA) leads to dispersal of mitochondrial fragments throughout the cytosol. The cristae of these fragmented mitochondria are disorganized. At early time points after transfection with Mgm1/OPA1 siRNA, the mitochondria are not yet fragmented. Instead, the mitochondria swell and stretch, after which they form localized constrictions similar to the mitochondrial abnormalities observed during the early stages of apoptosis. These abnormalities might be the earliest effects of losing Mgm1/OPA1 protein.


Assuntos
GTP Fosfo-Hidrolases/fisiologia , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/fisiologia , Dilatação Mitocondrial , Fracionamento Celular , Microscopia Crioeletrônica , GTP Fosfo-Hidrolases/genética , Inativação Gênica , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólise , Membranas Intracelulares/química , Mitocôndrias/química , RNA Interferente Pequeno/farmacologia , Transfecção
6.
Mol Biol Cell ; 14(4): 1583-96, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12686611

RESUMO

The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied in Saccharomyces cerevisiae. Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA. However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm. In this article we have used immunocytochemistry and biochemical isolation procedures to characterize the composition of human mitochondrial nucleoids. The results show that both the mitochondrial transcription factor TFAM and mitochondrial single-stranded DNA-binding protein colocalize with Twinkle in intramitochondrial foci defined as nucleoids by the specific incorporation of bromodeoxyuridine. Furthermore, mtDNA polymerase POLG and various other as yet unidentified proteins copurify with mtDNA nucleoids using a biochemical isolation procedure, as does TFAM. The results demonstrated that mtDNA in mammalian cells is organized in discrete protein-rich structures within the mitochondrial network. In vivo time-lapse imaging of nucleoids show they are dynamic structures able to divide and redistribute in the mitochondrial network and suggest that nucleoids are the mitochondrial units of inheritance. Nucleoids did not colocalize with dynamin-related protein 1, Drp1, a protein of the mitochondrial fission machinery.


Assuntos
DNA Mitocondrial/metabolismo , Proteínas Mitocondriais , Partículas Submitocôndricas/metabolismo , Sequência de Bases , Linhagem Celular , DNA Helicases , DNA Polimerase gama , DNA Primase/metabolismo , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
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