RESUMO
Mitochondrial ATP-synthesis is catalyzed by a F1Fo-ATP synthase, an enzyme of dual genetic origin enriched at the edge of cristae where it plays a key role in their structure/stability. The enzyme's biogenesis remains poorly understood, both from a mechanistic and a compartmentalization point of view. The present study provides novel molecular insights into this process through investigations on a human protein called TMEM70 with an unclear role in the assembly of ATP synthase. A recent study has revealed the existence of physical interactions between TMEM70 and the subunit c (Su.c), a protein present in 8 identical copies forming a transmembrane oligomeric ring (c-ring) within the ATP synthase proton translocating domain (Fo). Herein we analyzed the ATP-synthase assembly in cells lacking TMEM70, mitochondrial DNA or F1 subunits and observe a direct correlation between TMEM70 and Su.c levels, regardless of the status of other ATP synthase subunits or of mitochondrial bioenergetics. Immunoprecipitation, two-dimensional blue-native/SDS-PAGE, and pulse-chase experiments reveal that TMEM70 forms large oligomers that interact with Su.c not yet incorporated into ATP synthase complexes. Moreover, discrete TMEM70-Su.c complexes with increasing Su.c contents can be detected, suggesting a role for TMEM70 oligomers in the gradual assembly of the c-ring. Furthermore, we demonstrate using expansion super-resolution microscopy the specific localization of TMEM70 at the inner cristae membrane, distinct from the MICOS component MIC60. Taken together, our results show that TMEM70 oligomers provide a scaffold for c-ring assembly and that mammalian ATP synthase is assembled within inner cristae membranes.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Linhagem Celular , Metabolismo Energético , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana/genética , Microscopia Eletrônica , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Domínios Proteicos , Multimerização ProteicaRESUMO
Heme (iron protoporphyrin IX) is a well-known prosthetic group for enzymes involved in metabolic pathways such as oxygen transport and electron transfer through the mitochondrial respiratory chain. However, heme has also been shown to be an important regulatory molecule (as "labile" heme) for diverse processes such as translation, kinase activity, and transcription in mammals, yeast, and bacteria. Taking advantage of a yeast strain deficient for heme production that enabled controlled modulation and monitoring of labile heme levels, here we investigated the role of labile heme in the regulation of mitochondrial biogenesis. This process is regulated by the HAP complex in yeast. Using several biochemical assays along with EM and epifluorescence microscopy, to the best of our knowledge, we show for the first time that cellular labile heme is critical for the post-translational regulation of HAP complex activity, most likely through the stability of the transcriptional co-activator Hap4p. Consequently, we found that labile heme regulates mitochondrial biogenesis and cell growth. The findings of our work highlight a new mechanism in the regulation of mitochondrial biogenesis by cellular metabolites.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Hemina/metabolismo , Mitocôndrias/metabolismo , Biogênese de Organelas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator de Ligação a CCAAT/genética , Consumo de Oxigênio , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de SinaisRESUMO
Mitochondrial diseases are systemic, prevalent and often fatal; yet treatments remain scarce. Identifying molecular intervention points that can be therapeutically targeted remains a major challenge, which we confronted via a screening assay we developed. Using yeast models of mitochondrial ATP synthase disorders, we screened a drug repurposing library, and applied genomic and biochemical techniques to identify pathways of interest. Here we demonstrate that modulating the sorting of nuclear-encoded proteins into mitochondria, mediated by the TIM23 complex, proves therapeutic in both yeast and patient-derived cells exhibiting ATP synthase deficiency. Targeting TIM23-dependent protein sorting improves an array of phenotypes associated with ATP synthase disorders, including biogenesis and activity of the oxidative phosphorylation machinery. Our study establishes mitochondrial protein sorting as an intervention point for ATP synthase disorders, and because of the central role of this pathway in mitochondrial biogenesis, it holds broad value for the treatment of mitochondrial diseases.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Antifúngicos/farmacologia , Núcleo Celular/metabolismo , Bases de Dados de Produtos Farmacêuticos , Reposicionamento de Medicamentos , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/deficiência , Terapia de Alvo Molecular , Mutação , Proteínas Nucleares/genética , Fosforilação Oxidativa/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Piridinas/farmacologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Tionas/farmacologiaRESUMO
Mitochondria are double membrane-bounded organelles that form a dynamic tubular network. Mitochondria energetic functions depend on a complex internal architecture. Cristae, inner membrane invaginations that fold into the matrix space, are proposed to be the site of oxidative phosphorylation, reactions by which ATP synthase produces ATP. ATP synthase is also thought to have a role in crista morphogenesis. To date, the exploration of the processes regulating mitochondrial internal compartmentalization have been mostly limited to electron microscopy. Here, we describe ATP synthase localization in living yeast cells and show that it clusters as discrete inner membrane domains. These domains are dynamic within the mitochondrial network. They are impaired in mutants defective in crista morphology and partially overlap with the crista-associated MICOS-MINOS-MITOS complex. Finally, ATP synthase occupancy increases with the cellular demand for OXPHOS. Overall our data suggest that domains in which ATP synthases are clustered correspond to mitochondrial cristae. Being able to follow mitochondrial sub-compartments in living yeast cells opens new avenues to explore the mechanisms involved in inner membrane remodeling, an architectural feature crucial for mitochondrial activities.
Assuntos
Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fosforilação Oxidativa , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Metabolismo dos Carboidratos , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Mitocôndrias/ultraestrutura , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Imagem com Lapso de TempoRESUMO
Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA). We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS) due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP) or to maternally inherited Leigh Syndrome (MILS) in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from the network of functional, fusogenic mitochondria.
Assuntos
DNA Mitocondrial , Fusão de Membrana , Membranas Mitocondriais/metabolismo , Mutação , Proteínas de Ligação ao GTP/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fosforilação Oxidativa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mitochondrial dysfunction is linked to apoptosis, aging, cancer, and a number of neurodegenerative and muscular disorders. The interplay between mitophagy and mitochondrial dynamics has been linked to the removal of dysfunctional mitochondria ensuring mitochondrial quality control. An open question is what role mitochondrial fission plays in the removal of mitochondria after mild and transient oxidative stress; conditions reported to result in moderately elevated reactive oxygen species (ROS) levels comparable to physical activity. Here we show that applying such conditions led to fragmentation of mitochondria and induction of mitophagy in mouse and human cells. These conditions increased ROS levels only slightly and neither triggered cell death nor led to a detectable induction of non-selective autophagy. Starvation led to hyperfusion of mitochondria, to high ROS levels, and to the induction of both non-selective autophagy and to a lesser extent to mitophagy. We conclude that moderate levels of ROS specifically trigger mitophagy but are insufficient to trigger non-selective autophagy. Expression of a dominant-negative variant of the fission factor DRP1 blocked mitophagy induction by mild oxidative stress as well as by starvation. Taken together, we demonstrate that in mammalian cells under mild oxidative stress a DRP1-dependent type of mitophagy is triggered while a concomitant induction of non-selective autophagy was not observed. We propose that these mild oxidative conditions resembling well physiological situations are thus very helpful for studying the molecular pathways governing the selective removal of dysfunctional mitochondria.
Assuntos
Autofagia , Mitocôndrias/patologia , Dinâmica Mitocondrial/fisiologia , Mitofagia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Proteína 5 Relacionada à Autofagia , Western Blotting , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/fisiologia , Mitocôndrias/metabolismoRESUMO
In yeast, the two main F(O) proton-translocating subunits of the ATP synthase (subunits 6/a and 9/c) are encoded by mitochondrial DNA (mtDNA). Unfortunately, mutations that inactivate the F(O) typically result in loss of mtDNA under the form of ρ(-)/ρ(0) cells. Thus, we have designed a novel genetic strategy to circumvent this problem. It exploits previous findings that a null mutation in the nuclear ATP16 gene encoding ATP synthase subunit δ results in massive and lethal F(O)-mediated protons leaks across the inner mitochondrial membrane. Mutations that inactivate the F(O) can thus, in these conditions, be selected positively as cell viability rescuing events. A first set of seven mutants was analyzed and all showed, as expected, very severe F(O) deficiencies. Two mutants carried nuclear mutations in known genes (AEP1, AEP2) required for subunit c expression. The five other mutations were located in mtDNA. Of these, three affect synthesis or stability of subunit a transcripts and the two last consisted in a single amino acid replacement in subunit c. One of the subunit c mutations is particularly interesting. It consists in an alanine to valine change at position 60 of subunit c adjacent to the essential glutamate of subunit c (at position 59) that interacts with the essential arginine 186 of subunit a. The properties of this mutant suggest that the contact zone between subunit a and the ten subunits c-ring structure only involves critical transient interactions confined to the region where protons are exchanged between the subunit a and the c-ring.
Assuntos
Mutação , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Domínio Catalítico , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Rhomboids are a family of intramembrane serine proteases that are conserved in bacteria, archaea, and eukaryotes. They are required for numerous fundamental cellular functions such as quorum sensing, cell signaling, and mitochondrial dynamics. Mitochondrial rhomboids form an evolutionarily distinct class of rhomboids. It is largely unclear how their activity is controlled and which substrate determinants are responsible for recognition and cleavage. We investigated these requirements for the mitochondrial rhomboid protease Pcp1 and its substrate Mgm1. In contrast to several other rhomboid proteases, Pcp1 does not require helix-breaking amino acids in the cleaved hydrophobic region of Mgm1, termed 'rhomboid cleavage region' (RCR). Even transmembrane segments of inner membrane proteins that are normally not processed by Pcp1 become cleavable when put in place of the authentic RCR of Mgm1. We further show that mutational alterations of a highly negatively charged region located C-terminally to the RCR led to a strong processing defect. Moreover, we show that the determinants required for Mgm1 processing by mitochondrial rhomboid protease are conserved during evolution, as PARL (the human ortholog of Pcp1) showed similar substrate requirements. These results suggest a surprising promiscuity of the mitochondrial rhomboid protease regarding the sequence requirements of the cleaved hydrophobic segment. We propose a working hypothesis on how the mitochondrial rhomboid protease can, despite this promiscuity, achieve a high specificity in recognizing Mgm1. This hypothesis relates to the exceptional biogenesis pathway of Mgm1.
Assuntos
Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina Endopeptidases/metabolismo , Serina Proteases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Sequência Conservada , Proteínas de Ligação ao GTP/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/metabolismo , Metaloproteases/metabolismo , Proteínas Mitocondriais/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Mitochondria are the site where oxidative phosphorylations (OXPHOSs) take place. Fusion and fission reactions allow them to change their overall morphology, which ranges from networks of elongated and branched filaments to collections of small individual organelles. It is assumed that mitochondrial bioenergetics and dynamics are linked and that mitochondrial morphology reflects their functional status. This review shows that the links between mitochondrial dynamics and bioenergetics are complex and that mitochondrial deficiencies are not systematically associated to fragmentation. In mammals, mitochondrial fragmentation is observed upon inhibition of OXPHOS with drugs, but not in most cellular models with OXPHOS deficits of genetic origin. In yeast, mitochondrial biogenesis and filament interconnectivity augment with increasing respiratory capacity, but mutation or inhibition of the respiratory chain does not provoke major morphological changes. Significant structural and morphological alterations appear restricted to mutation of genes involved in assembly or function of the F(1)F(0)-ATP-synthase. Finally, ex vivo studies (in mammals) and in vitro studies (in yeast) confirm the essential role of the inner membrane potential for mitochondrial fusion.
Assuntos
Metabolismo Energético , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Animais , Humanos , Fusão de Membrana , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fosforilação OxidativaRESUMO
The mitochondrial dynamin-like GTPase Mgm1 exists as a long (l-Mgm1) and a short isoform (s-Mgm1). They both are essential for mitochondrial fusion. Here we show that the isoforms interact in a homotypic and heterotypic manner. Their submitochondrial distribution between inner boundary membrane and cristae was markedly different. Overexpression of l-Mgm1 exerts a dominant negative effect on mitochondrial fusion. A functional GTPase domain is required only in s-Mgm1 but not in l-Mgm1. We propose that l-Mgm1 acts primarily as an anchor in the inner membrane that in concert with the GTPase activity of s-Mgm1 mediates the fusion of inner membranes.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Dinaminas/química , Dinaminas/metabolismo , Microscopia Imunoeletrônica , Proteínas Mitocondriais/genética , Isoformas de Proteínas/metabolismoRESUMO
Mitochondrial genomes generally encode a minimal set of tRNAs necessary for protein synthesis. However, a number of eukaryotes import tRNAs from the cytoplasm into their mitochondria. For instance, Saccharomyces cerevisiae imports cytoplasmic tRNA(Gln) into the mitochondrion without any added protein factors. Here, we examine the existence of a similar active tRNA import system in mammalian mitochondria. We have used subcellular RNA fractions from rat liver and human cells to perform RT-PCR with oligonucleotide primers specific for nucleus-encoded tRNA(CUG)(Gln) and tRNA(UUG)(Gln) species, and we show that these tRNAs are present in rat and human mitochondria in vivo. Import of in vitro transcribed tRNAs, but not of heterologous RNAs, into isolated mitochondria also demonstrates that this process is tRNA-specific and does not require the addition of cytosolic factors. Although this in vitro system requires ATP, it is resistant to inhibitors of the mitochondrial electrochemical gradient, a key component of protein import. tRNA(Gln) import into mammalian mitochondria proceeds by a mechanism distinct from protein import. We also show that both tRNA(Gln) species and a bacterial pre-tRNA(Asp) can be imported in vitro into mitochondria isolated from myoclonic epilepsy with ragged-red fiber cells if provided with sufficient ATP (2 mM). This work suggests that tRNA import is more widespread than previously thought and may be a universal trait of mitochondria. Mutations in mitochondrial tRNA genes have been associated with human disease; the tRNA import system described here could possibly be exploited for the manipulation of defective mitochondria.
Assuntos
Mamíferos/metabolismo , Mitocôndrias/metabolismo , Transporte de RNA , RNA de Transferência de Glutamina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sequência de Bases , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Células HeLa , Humanos , Síndrome MERRF/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte de RNA/efeitos dos fármacos , RNA de Transferência de Glutamina/química , RNA de Transferência de Glutamina/genética , Ratos , Solubilidade/efeitos dos fármacosRESUMO
NARP (neuropathy, ataxia, and retinitis pigmentosa) and MILS (maternally inherited Leigh syndrome) are mitochondrial disorders associated with point mutations of the mitochondrial DNA (mtDNA) in the gene encoding the Atp6p subunit of the ATP synthase. The most common and studied of these mutations is T8993G converting the highly conserved leucine 156 into arginine. We have introduced this mutation at the corresponding position (183) of yeast Saccharomyces cerevisiae mitochondrially encoded Atp6p. The "yeast NARP mutant" grew very slowly on respiratory substrates, possibly because mitochondrial ATP synthesis was only 10% of the wild type level. The mutated ATP synthase was found to be correctly assembled and present at nearly normal levels (80% of the wild type). Contrary to what has been reported for human NARP cells, the reverse functioning of the ATP synthase, i.e. ATP hydrolysis in the F(1) coupled to F(0)-mediated proton translocation out of the mitochondrial matrix, was significantly compromised in the yeast NARP mutant. Interestingly, the oxygen consumption rate in the yeast NARP mutant was decreased by about 80% compared with the wild type, due to a selective lowering in cytochrome c oxidase (complex IV) content. This finding suggests a possible regulatory mechanism between ATP synthase activity and complex IV expression in yeast mitochondria. The availability of a yeast NARP model could ease the search for rescuing mechanisms against this mitochondrial disease.
Assuntos
Ataxia/enzimologia , Doença de Leigh/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Biológicos , Retinose Pigmentar/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/genética , Substituição de Aminoácidos , Ataxia/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidrólise , Mitocôndrias/enzimologia , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação de Sentido Incorreto , Consumo de Oxigênio/genética , Prótons , Retinose Pigmentar/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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 SubstratoRESUMO
Atp6p is an essential subunit of the ATP synthase proton translocating domain, which is encoded by the mitochondrial DNA (mtDNA) in yeast. We have replaced the coding sequence of Atp6p gene with the non-respiratory genetic marker ARG8m. Due to the presence of ARG8m, accumulation of rho-/rho0 petites issued from large deletions in mtDNA could be restricted to 20-30% by growing the atp6 mutant in media lacking arginine. This moderate mtDNA instability created favorable conditions to investigate the consequences of a specific lack in Atp6p. Interestingly, in addition to the expected loss of ATP synthase activity, the cytochrome c oxidase respiratory enzyme steady-state level was found to be extremely low (<5%) in the atp6 mutant. We show that the cytochrome c oxidase-poor accumulation was caused by a failure in the synthesis of one of its mtDNA-encoded subunits, Cox1p, indicating that, in yeast mitochondria, Cox1p synthesis is a key target for cytochrome c oxidase abundance regulation in relation to the ATP synthase activity. We provide direct evidence showing that in the absence of Atp6p the remaining subunits of the ATP synthase can still assemble. Mitochondrial cristae were detected in the atp6 mutant, showing that neither Atp6p nor the ATP synthase activity is critical for their formation. However, the atp6 mutant exhibited unusual mitochondrial structure and distribution anomalies, presumably caused by a strong delay in inner membrane fusion.
Assuntos
Genes Mitocondriais/genética , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Forma Celular , Citocromos c/metabolismo , DNA Mitocondrial/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genótipo , Hidrólise , Microscopia Eletrônica , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Oxirredutases/metabolismo , Oxigênio/metabolismo , Biossíntese de Proteínas/genética , Saccharomyces cerevisiae/genética , Transaminases/genética , Transaminases/metabolismo , Transcrição Gênica/genéticaRESUMO
Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase gamma. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network.
Assuntos
GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Animais , Estudos de Casos e Controles , Linhagem Celular , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Metabolismo Energético , Células HeLa , Proteínas de Grupo de Alta Mobilidade/deficiência , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Mutação , Processamento de Proteína Pós-TraducionalRESUMO
Mitochondrial diseases are rare diseases most often linked to energy in the form of ATP-depletion. The high number of nuclear- and mitochondrial-DNA-encoded proteins (>500), required for ATP production and other crucial mitochondrial functions such as NADH re-oxidation, explains the increasing number of reported disorders. In recent years, yeast has revealed to be a powerful model to identify responsible genes, to study primary effects of pathogenic mutations and to determine the molecular mechanisms leading to mitochondrial disorders. However, the clinical management of patients with mitochondrial disorders is still essentially supportive. Here we review some of the most fruitful yeast mitochondrial disorder models and propose to subject these models to highthroughput chemical library screening to prospect new therapeutic drugs against mitochondrial diseases.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Mitocôndrias/metabolismo , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Animais , Bioensaio/métodos , Sistemas de Liberação de Medicamentos/métodos , Desenho de Fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Biologia Molecular/métodosRESUMO
Within the mitochondrial F(1)F(0)-ATP synthase, the nucleus-encoded delta-F(1) subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the delta subunit gene (Deltadelta) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% rho(-)/rho degrees petites (i.e. cells missing a large portion (>50%) of the mtDNA (rho(-)) or totally devoid of mtDNA (rho degrees )). Previous work has suggested that the absence of complete mtDNA (rho(+)) in Deltadelta yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8(m)) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Deltadelta yeast. We further show that combined with a nuclear mutation (Deltaatp4) preventing the ATP synthase proton channel assembly, a lack of delta subunit fails to destabilize the mtDNA, and rho(+) Deltadelta cells become viable. We conclude that Deltadelta yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the rho(-)/rho degrees mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae.
Assuntos
DNA Mitocondrial , Genes Fúngicos , Mitocôndrias/enzimologia , Mutação , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Transporte Biológico , Regulação Fúngica da Expressão Gênica , ATPases Mitocondriais Próton-Translocadoras/genética , Modelos Biológicos , Modelos Genéticos , Prótons , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The mitochondrial ATP synthase is made of a membrane-integrated F0 component that forms a proton-permeable pore through the inner membrane and a globular peripheral F1 domain where ATP is synthesized. The catalytic mechanism is thought to involve the rotation of a 10-12 c subunit ring in the F0 together with the gamma subunit of F1. An important and not yet resolved question is to define precisely how the gamma subunit is connected with the c-ring. In this study, using a doxycycline-regulatable expression system, we provide direct evidence that the rest of the enzyme can assemble without the delta subunit of F1, and we show that delta-less mitochondria are uncoupled because of an F0-mediated proton leak. Based on these observations, and taking into account high-resolution structural models, we propose that subunit delta plays a key role in the mechanical coupling of the c-ring to subunit gamma.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/química , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/metabolismo , Catálise , Membrana Celular/metabolismo , Doxiciclina/farmacologia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Hidrólise , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Consumo de Oxigênio , Regiões Promotoras Genéticas , Conformação Proteica , Estrutura Terciária de Proteína , ATPases Translocadoras de Prótons/química , Fatores de TempoRESUMO
Mutant strains of yeast Saccharomyces cerevisiae lacking a functional F1-ATPase were found to grow very poorly under anaerobic conditions. A single amino acid replacement (K222 > E222) that locally disrupts the adenine nucleotide catalytic site in the beta-F1 subunit was sufficient to compromise anaerobic growth. This mutation also affected growth in aerated conditions when ethidium bromide (an intercalating agent impairing mtDNA propagation) or antimycin (an inhibitor of respiration) was included in the medium. F1-deficient cells forced to grow in oxygen-limited conditions were shown to lose their mtDNA completely and to accumulate Hsp60p mainly under its precursor form. Fluorescence microscopy analyses with a modified GFP containing a mitochondrial targeting presequence revealed that aerobically growing F1-deficient cells stopped importing the GFP when antimycin was added to the medium. Finally, after total inactivation of the catalytic alpha3beta3 subcomplex of F1, mitochondria could no longer be energized by externally added ATP because of either a block in assembly or local disruption of the adenine nucleotide processing site. Altogether these data strengthen the notion that in the absence of respiration, and whether the proton translocating domain (F0) of complex V is present or not, F1-catalysed hydrolysis of ATP is essential for the occurrence of vital cellular processes depending on the maintenance of an electrochemical potential across the mitochondrial inner membrane.
Assuntos
Trifosfato de Adenosina/metabolismo , Antimicina A/análogos & derivados , Mitocôndrias/fisiologia , ATPases Translocadoras de Prótons/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Substituição de Aminoácidos , Anaerobiose , Antimicina A/farmacologia , Sítios de Ligação , Hipóxia Celular , DNA Mitocondrial/biossíntese , Genes Reporter , Proteínas de Fluorescência Verde , Hidrólise , Membranas Intracelulares/fisiologia , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Potenciais da Membrana , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Fosforilação Oxidativa , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas , Bombas de Próton/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Proteínas Recombinantes de Fusão/análise , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The [Het-s] infectious element of the filamentous fungus Podospora anserina is a prion. We have recently reported that recombinant HET-s protein aggregates in vitro into amyloid fibers. In vivo, the protein aggregates specifically in the [Het-s] prion strains. Here, we show that biolistic introduction of aggregated recombinant HET-s protein into fungal cells induces emergence of the [Het-s] prion with a high frequency. Thus, we demonstrate that prion infectivity can be created de novo, in vitro from recombinant protein in this system. Although the amyloid filaments formed from HET-s could transmit [Het-s] efficiently, neither the soluble form of the protein nor amorphous aggregates would do so. In addition, we have found that (i) [Het-s] infectivity correlates with the ability to convert HET-s to amyloids in vitro, (ii) [Het-s] infectivity is resistant to proteinase K digestion, and (iii) HET-s aggregates formed in vivo in [Het-s] strains have the ability to convert the recombinant protein to aggregates. Together, our data designate the HET-s amyloids as the molecular basis of [Het-s] prion propagation.