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1.
PLoS Genet ; 8(8): e1002876, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22916027

RESUMO

Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms.


Assuntos
Núcleo Celular/genética , Proteínas Fúngicas/genética , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Podospora/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Evolução Biológica , Núcleo Celular/enzimologia , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes Mitocondriais , Genoma Mitocondrial , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fosforilação Oxidativa , Podospora/enzimologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Transgenes
2.
Mol Biol Evol ; 28(7): 2063-75, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21273631

RESUMO

An F(1)F(O) ATP synthase in the inner mitochondrial membrane catalyzes the late steps of ATP production via the process of oxidative phosphorylation. A small protein subunit (subunit c or ATP9) of this enzyme shows a substantial genetic diversity, and its gene can be found in both the mitochondrion and/or nucleus. In a representative set of 26 species of fungi for which the genomes have been entirely sequenced, we found five Atp9 gene repartitions. The phylogenetic distribution of nuclear and mitochondrial Atp9 genes suggests that their evolution has included two independent transfers to the nucleus followed by several independent episodes of the loss of the mitochondrial and/or nuclear gene. Interestingly, we found that in Podospora anserina, subunit c is exclusively produced from two nuclear genes (PaAtp9-5 and PaAtp9-7), which display different expression profiles through the life cycle of the fungus. The PaAtp9-5 gene is specifically and strongly expressed in germinating ascospores, whereas PaAtp9-7 is mostly transcribed during sexual reproduction. Consistent with these observations, deletion of PaAtp9-5 is lethal, whereas PaAtp9-7 deletion strongly impairs ascospore production. The P. anserina PaAtp9-5 and PaAtp9-7 genes are therefore nonredundant. By swapping the 5' and 3' flanking regions between genes we demonstrated, however, that the PaAtp9 coding sequences are functionally interchangeable. These findings show that after transfer to the nucleus, the subunit c gene in Podospora became a key target for the modulation of cellular energy metabolism according to the requirements of the life cycle.


Assuntos
Proteínas Fúngicas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , ATPases Mitocondriais Próton-Translocadoras/genética , Podospora/genética , Sequência de Bases , Núcleo Celular , Proteínas Fúngicas/metabolismo , Deleção de Genes , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Dados de Sequência Molecular , Micélio/genética , Micélio/crescimento & desenvolvimento , Fenótipo , Filogenia , Podospora/enzimologia , Podospora/crescimento & desenvolvimento , Subunidades Proteicas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento
3.
Genetics ; 166(1): 135-50, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15020413

RESUMO

It has been previously reported that, at the time of death, the Podospora anserina AS1-4 mutant strains accumulate specific deleted forms of the mitochondrial genome and that their life spans depend on two natural alleles (variants) of the rmp1 gene: AS1-4 rmp1-2 strains exhibit life spans strikingly longer than those of AS1-4 rmp1-1. Here, we show that rmp1 is an essential gene. In silico analyses of eight rmp1 natural alleles present in Podospora isolates and of the putative homologs of this orphan gene in other filamentous fungi suggest that rmp1 evolves rapidly. The RMP1 protein is localized in the mitochondrial and/or the cytosolic compartment, depending on cell type and developmental stage. Strains producing RMP1 without its mitochondrial targeting peptide are viable but exhibit vegetative and sexual defects.


Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Sordariales/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Núcleo Celular/metabolismo , Clonagem Molecular , DNA Fúngico/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Mutação , Polimorfismo Genético , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Sordariales/crescimento & desenvolvimento , Sordariales/metabolismo
4.
PLoS One ; 7(5): e38138, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22693589

RESUMO

Mitochondria have their own ATP-dependent proteases that maintain the functional state of the organelle. All multicellular eukaryotes, including filamentous fungi, possess the same set of mitochondrial proteases, unlike in unicellular yeasts, where ClpXP, one of the two matricial proteases, is absent. Despite the presence of ClpXP in the filamentous fungus Podospora anserina, deletion of the gene encoding the other matricial protease, PaLon1, leads to lethality at high and low temperatures, indicating that PaLON1 plays a main role in protein quality control. Under normal physiological conditions, the PaLon1 deletion is viable but decreases life span. PaLon1 deletion also leads to defects in two steps during development, ascospore germination and sexual reproduction, which suggests that PaLON1 ensures important regulatory functions during fungal development. Mitochondrial Lon proteases are composed of a central ATPase domain flanked by a large non-catalytic N-domain and a C-terminal protease domain. We found that three mutations in the N-domain of PaLON1 affected fungal life cycle, PaLON1 protein expression and mitochondrial proteolytic activity, which reveals the functional importance of the N-domain of the mitochondrial Lon protease. All PaLon1 mutations affected the C-terminal part of the N-domain. Considering that the C-terminal part is predicted to have an α helical arrangement in which the number, length and position of the helices are conserved with the solved structure of its bacterial homologs, we propose that this all-helical structure participates in Lon substrate interaction.


Assuntos
Mitocôndrias/enzimologia , Podospora/citologia , Podospora/enzimologia , Protease La/química , Protease La/metabolismo , Alelos , Sequência de Aminoácidos , Deleção de Genes , Dados de Sequência Molecular , Fenótipo , Podospora/genética , Podospora/fisiologia , Protease La/deficiência , Protease La/genética , Estrutura Terciária de Proteína , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/genética
5.
Genome Biol ; 9(5): R77, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18460219

RESUMO

BACKGROUND: The dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development. RESULTS: We present a 10X draft sequence of P. anserina genome, linked to the sequences of a large expressed sequence tag collection. Similar to higher eukaryotes, the P. anserina transcription/splicing machinery generates numerous non-conventional transcripts. Comparison of the P. anserina genome and orthologous gene set with the one of its close relatives, Neurospora crassa, shows that synteny is poorly conserved, the main result of evolution being gene shuffling in the same chromosome. The P. anserina genome contains fewer repeated sequences and has evolved new genes by duplication since its separation from N. crassa, despite the presence of the repeat induced point mutation mechanism that mutates duplicated sequences. We also provide evidence that frequent gene loss took place in the lineages leading to P. anserina and N. crassa. P. anserina contains a large and highly specialized set of genes involved in utilization of natural carbon sources commonly found in its natural biotope. It includes genes potentially involved in lignin degradation and efficient cellulose breakdown. CONCLUSION: The features of the P. anserina genome indicate a highly dynamic evolution since the divergence of P. anserina and N. crassa, leading to the ability of the former to use specific complex carbon sources that match its needs in its natural biotope.


Assuntos
Evolução Molecular , Genoma Fúngico , Podospora/genética , Sequência de Bases , Carbono/metabolismo , Etiquetas de Sequências Expressas , Duplicação Gênica , Dados de Sequência Molecular , Neurospora crassa/genética , Podospora/metabolismo
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