RESUMO
The endoplasmic reticulum (ER) is an elaborate organelle composed of distinct structural and functional domains. ER structure and dynamics involve membrane-shaping proteins of the reticulon and Yop1/DP1 families, which promote membrane curvature and regulate ER shaping and remodeling. Here, we analyzed the function of the reticulon (RTN1) and Yop1 proteins (YOP1 and YOP2) of the model fungus Podospora anserina and their contribution to sexual development. We found that RTN1 and YOP2 localize to the peripheral ER and are enriched in the dynamic apical ER domains of the polarized growing hyphal region. We discovered that the formation of these domains is diminished in the absence of RTN1 or YOP2 and abolished in the absence of YOP1 and that hyphal growth is moderately reduced when YOP1 is deleted in combination with RTN1 and/or YOP2. In addition, we found that RTN1 associates with the Spitzenkörper. Moreover, RTN1 localization is regulated during meiotic development, where it accumulates at the apex of growing asci (meiocytes) during their differentiation and at their middle region during the subsequent meiotic progression. Furthermore, we discovered that loss of RTN1 affects ascospore (meiotic spore) formation, in a process that does not involve YOP1 or YOP2. Finally, we show that the defects in ascospore formation of rtn1 mutants are associated with defective nuclear segregation and spindle dynamics throughout meiotic development. Our results show that sexual development in P. anserina involves a developmental remodeling of the ER that implicates the reticulon RTN1, which is required for meiotic nucleus segregation. IMPORTANCE Meiosis consists of a reductional cell division, which allows ploidy maintenance during sexual reproduction and which provides the potential for genetic recombination, producing genetic variation. Meiosis constitutes a process of foremost importance for eukaryotic evolution. Proper partitioning of nuclei during this process relies on accurate functioning and positioning of the spindle, the microtubule cytoskeletal apparatus that conducts chromosome segregation. In this research, we show that in the model fungus Podospora anserina this process requires a protein involved in structuring the endoplasmic reticulum (ER)-the reticulon RTN1. The ER is a complex organelle composed of distinct structural domains, including different peripheral domains and the nuclear envelope. Our findings suggest that spindle dynamics during meiosis relies on remodeling of the ER membrane, which involves the activity of RTN1. Our research discloses that the proteins implicated in shaping the ER are main contributors to the regulation of nuclear dynamics during the sexual cycle.
Assuntos
Retículo Endoplasmático/metabolismo , Meiose , Podospora/genética , Podospora/fisiologia , Segregação de Cromossomos , Proteínas de Membrana/metabolismo , Microtúbulos , Membrana Nuclear , Podospora/citologia , Fuso Acromático/metabolismo , Esporos FúngicosRESUMO
Mitochondrial F1Fo-ATP-synthase dimers play a critical role in shaping and maintenance of mitochondrial ultrastructure. Previous studies have revealed that ablation of the F1Fo-ATP-synthase assembly factor PaATPE of the ascomycete Podospora anserina strongly affects cristae formation, increases hydrogen peroxide levels, impairs mitochondrial function and leads to premature cell death. In the present study, we investigated the underlying mechanistic basis. Compared to the wild type, we observed a slight increase in non-selective and a pronounced increase in mitophagy, the selective vacuolar degradation of mitochondria. This effect depends on the availability of functional cyclophilin D (PaCYPD), the regulator of the mitochondrial permeability transition pore (mPTP). Simultaneous deletion of PaAtpe and PaAtg1, encoding a key component of the autophagy machinery or of PaCypD, led to a reduction of mitophagy and a partial restoration of the wild-type specific lifespan. The same effect was observed in the PaAtpe deletion strain after inhibition of PaCYPD by its specific inhibitor, cyclosporin A. Overall, our data identify autophagy-dependent cell death (ADCD) as part of the cellular response to impaired F1Fo-ATP-synthase dimerization, and emphasize the crucial role of functional mitochondria in aging.
Assuntos
Morte Celular Autofágica , Peptidil-Prolil Isomerase F/metabolismo , Podospora/enzimologia , Podospora/fisiologia , Multimerização Proteica , ATPases Translocadoras de Prótons/metabolismo , Deleção de Genes , Peróxido de Hidrogênio/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Mitofagia , Podospora/citologia , ATPases Translocadoras de Prótons/deficiência , Vacúolos/metabolismoRESUMO
In plants and metazoans, intracellular receptors that belong to the NOD-like receptor (NLR) family are major contributors to innate immunity. Filamentous fungal genomes contain large repertoires of genes encoding for proteins with similar architecture to plant and animal NLRs with mostly unknown function. Here, we identify and molecularly characterize patatin-like phospholipase-1 (PLP-1), an NLR-like protein containing an N-terminal patatin-like phospholipase domain, a nucleotide-binding domain (NBD), and a C-terminal tetratricopeptide repeat (TPR) domain. PLP-1 guards the essential SNARE protein SEC-9; genetic differences at plp-1 and sec-9 function to trigger allorecognition and cell death in two distantly related fungal species, Neurospora crassa and Podospora anserina Analyses of Neurospora population samples revealed that plp-1 and sec-9 alleles are highly polymorphic, segregate into discrete haplotypes, and show transspecies polymorphism. Upon fusion between cells bearing incompatible sec-9 and plp-1 alleles, allorecognition and cell death are induced, which are dependent upon physical interaction between SEC-9 and PLP-1. The central NBD and patatin-like phospholipase activity of PLP-1 are essential for allorecognition and cell death, while the TPR domain and the polymorphic SNARE domain of SEC-9 function in conferring allelic specificity. Our data indicate that fungal NLR-like proteins function similar to NLR immune receptors in plants and animals, showing that NLRs are major contributors to innate immunity in plants and animals and for allorecognition in fungi.
Assuntos
Apoptose , Proteínas Fúngicas/metabolismo , Proteínas NLR/metabolismo , Neurospora crassa/metabolismo , Podospora/metabolismo , Proteínas SNARE/metabolismo , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Proteínas NLR/química , Proteínas NLR/genética , Neurospora crassa/química , Neurospora crassa/citologia , Neurospora crassa/genética , Podospora/química , Podospora/citologia , Podospora/genética , Ligação Proteica , Domínios Proteicos , Proteínas SNARE/química , Proteínas SNARE/genética , Alinhamento de SequênciaRESUMO
Peroxisomes are versatile organelles essential for diverse developmental processes. One such process is the meiotic development of Podospora anserina. In this fungus, absence of the docking peroxin PEX13, the RING-finger complex peroxins, or the PTS2 co-receptor PEX20 blocks sexual development before meiocyte formation. However, this defect is not seen in the absence of the receptors PEX5 and PEX7, or of the docking peroxins PEX14 and PEX14/17. Here we describe the function of the remaining uncharacterized P. anserina peroxins predictably involved in peroxisome matrix protein import. We show that PEX8, as well as the peroxins potentially mediating receptor monoubiquitination (PEX4 and PEX22) and membrane dislocation (PEX1, PEX6 and PEX26) are indeed implicated in peroxisome matrix protein import in this fungus. However, we observed that elimination of PEX4 and PEX22 affects to different extent the import of distinct PEX5 cargoes, suggesting differential ubiquitination-complex requirements for the import of distinct proteins. In addition, we found that elimination of PEX1, PEX6 or PEX26 results in loss of peroxisomes, suggesting that these peroxins restrain peroxisome removal in specific physiological conditions. Finally, we demonstrate that all analyzed peroxins are required for meiocyte formation, and that PEX20 function in this process depends on its potential monoubiquitination target cysteine. Our results suggest that meiotic induction relies on a peroxisome import pathway, which is not dependent on PEX5 or PEX7 but that is driven by an additional cycling receptor. These findings uncover a collection of peroxins implicated in modulating peroxisome activity to facilitate a critical developmental cell fate decision.
Assuntos
Proteínas Fúngicas/metabolismo , Meiose , Peroxissomos/metabolismo , Podospora/citologia , Podospora/metabolismo , Receptores de Superfície Celular/metabolismo , Cisteína/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Modelos Biológicos , Micélio/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , UbiquitinaçãoRESUMO
Mitochondrial dysfunction is causatively linked to organismal aging and the development of degenerative diseases. Here we describe stress-dependent opposing roles of mitophagy, the selective autophagic degradation of mitochondria, in aging and life-span control. We report that the ablation of the mitochondrial superoxide dismutase which is involved in reactive oxygen species (ROS) balancing, does not affect life span of the fungal aging model Podospora anserina, although superoxide levels are strongly increased and complex I-dependent respiration is impaired. This unexpected phenotype depends on functional autophagy, particularly mitophagy, which is upregulated during aging of this mutant. It identifies mitophagy as a prosurvival response involved in the control of mitohormesis, the well-known beneficial effect of mild mitochondrial oxidative stress. In contrast, excessive superoxide stress turns mitophagy to a prodeath pathway and leads to accelerated aging. Overall our data uncover mitophagy as a dynamic pathway that specifically responds to different levels of mitochondrial oxidative stress and thereby affects organismal aging.
Assuntos
Mitofagia , Podospora/metabolismo , Podospora/fisiologia , Estresse Fisiológico , Autofagia , Biomarcadores/metabolismo , Morte Celular , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Homeostase , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Mitofagia/genética , Modelos Biológicos , Oxirredução , Estresse Oxidativo , Fenótipo , Podospora/citologia , Podospora/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Estresse Fisiológico/genética , Superóxidos/metabolismo , Transcrição Gênica , Regulação para Cima/genéticaRESUMO
For biological systems, balancing cellular levels of reactive oxygen species (ROS) is of great importance because ROS are both, essential for cellular signaling and dangerous in causing molecular damage. Cellular ROS abundance is controlled by a delicate network of molecular pathways. Within this network, superoxide dismutases (SODs) are active in disproportion of the superoxide anion leading to the formation of hydrogen peroxide. The fungal aging model Podospora anserina encodes at least three SODs. One of these is the mitochondrial PaSOD3 isoform containing manganese as a cofactor. Previous work resulted in the selection of strains in which PaSod3 is strongly overexpressed. These strains display impairments in growth and lifespan. A computational model suggests a series of events to occur in Sod3 overexpressing strains leading to adverse effects due to elevated hydrogen peroxide levels. In an attempt to validate this model and to obtain more detailed information about the cellular responses involved in ROS balancing, we further investigated the PaSod3 overexpressing strains. Here we show that hydrogen peroxide levels are indeed strongly increased in the mutant strain. Surprisingly, this phenotype can be rescued by the addition of manganese to the growth medium. Strikingly, while we obtained no evidence for an antioxidant effect of manganese, we found that the metal is required for induction of components of the ROS scavenging network and lowers the hydrogen peroxide level of the mutant. A similar effect of manganese on lifespan reversion was obtained in wild-type strains challenged with exogenous hydrogen peroxide. It appears that manganese is limited under high hydrogen peroxide and suggests that a manganese-dependent activity leads to the induction of ROS scavenging components.
Assuntos
Peróxido de Hidrogênio/metabolismo , Manganês/farmacologia , Podospora/citologia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Genótipo , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Podospora/genéticaRESUMO
Pseudo-homothallism is a reproductive strategy elected by some fungi producing heterokaryotic sexual spores containing genetically different but sexually compatible nuclei. This lifestyle appears as a compromise between true homothallism (self-fertility with predominant inbreeding) and complete heterothallism (with exclusive outcrossing). However, pseudohomothallic species face the problem of maintaining heterokaryotic mycelia to fully benefit from this lifestyle, as homokaryons are self-sterile. Here, we report on the structure of chromosome 1 in mat+ and mat- isolates of strain S of the pseudohomothallic fungus Podospora anserina. Chromosome 1 contains either one of the mat+ and mat- mating types of P. anserina, which is mostly found in nature as a mat+/mat- heterokaryotic mycelium harboring sexually compatible nuclei. We identified a "mat" region â¼0.8 Mb long, devoid of meiotic recombination and containing the mating-type idiomorphs, which is a candidate to be involved in the maintenance of the heterokaryotic state, since the S mat+ and S mat- strains have different physiology that may enable hybrid-vigor-like phenomena in the heterokaryons. The mat region contains 229 coding sequences. A total of 687 polymorphisms were detected between the S mat+ and S mat- chromosomes. Importantly, the mat region is colinear between both chromosomes, which calls for an original mechanism of recombination inhibition. Microarray analyses revealed that 10% of the P. anserina genes have different transcriptional profiles in S mat+ and S mat-, in line with their different phenotypes. Finally, we show that the heterokaryotic state is faithfully maintained during mycelium growth of P. anserina, yet mat+/mat+ and mat-/mat- heterokaryons are as stable as mat+/mat- ones, evidencing a maintenance of heterokaryosis that does not rely on fitness-enhancing complementation between the S mat+ and S mat- strains.
Assuntos
Núcleo Celular/genética , Loci Gênicos/genética , Podospora/genética , Podospora/fisiologia , Centrômero/genética , Cromossomos Fúngicos/genética , Genes Fúngicos/genética , Aptidão Genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Podospora/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética , Reprodução/genéticaRESUMO
Podospora anserina is an extensively studied model organism to unravel the mechanism of organismal aging. This filamentous fungus is short-lived and accessible to experimentation. Aging and lifespan are controlled by genetic and environmental traits and, in this model, have a strong mitochondrial etiology. Here, we describe methods and protocols to manipulate and study the aging process in P. anserina at different levels including biochemistry, cell biology, genetics, and physiology.
Assuntos
Podospora , Catalase/metabolismo , Fertilidade , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Viabilidade Microbiana , Mitocôndrias/metabolismo , Micélio/citologia , Micélio/genética , Micélio/metabolismo , Micélio/fisiologia , Oxigênio/metabolismo , Fenótipo , Podospora/citologia , Podospora/genética , Podospora/metabolismo , Podospora/fisiologia , RNA Fúngico/isolamento & purificação , RNA Fúngico/metabolismo , Esporos Fúngicos/citologia , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Esporos Fúngicos/fisiologia , Estresse Fisiológico , Fatores de Tempo , Transformação GenéticaRESUMO
The regulation of cellular copper homeostasis is crucial in biology. Impairments lead to severe dysfunctions and are known to affect aging and development. Previously, a loss-of-function mutation in the gene encoding the copper-sensing and copper-regulated transcription factor GRISEA of the filamentous fungus Podospora anserina was reported to lead to cellular copper depletion and a pleiotropic phenotype with hypopigmentation of the mycelium and the ascospores, affected fertility and increased lifespan by approximately 60% when compared to the wild type. This phenotype is linked to a switch from a copper-dependent standard to an alternative respiration leading to both a reduced generation of reactive oxygen species (ROS) and of adenosine triphosphate (ATP). We performed a genome-wide comparative transcriptome analysis of a wild-type strain and the copper-depleted grisea mutant. We unambiguously assigned 9,700 sequences of the transcriptome in both strains to the more than 10,600 predicted and annotated open reading frames of the P. anserina genome indicating 90% coverage of the transcriptome. 4,752 of the transcripts differed significantly in abundance with 1,156 transcripts differing at least 3-fold. Selected genes were investigated by qRT-PCR analyses. Apart from this general characterization we analyzed the data with special emphasis on molecular pathways related to the grisea mutation taking advantage of the available complete genomic sequence of P. anserina. This analysis verified but also corrected conclusions from earlier data obtained by single gene analysis, identified new candidates of factors as part of the cellular copper homeostasis system including target genes of transcription factor GRISEA, and provides a rich reference source of quantitative data for further in detail investigations. Overall, the present study demonstrates the importance of systems biology approaches also in cases were mutations in single genes are analyzed to explain the underlying mechanisms controlling complex biological processes like aging and development.
Assuntos
Fenômenos Biológicos/genética , Cobre/farmacologia , Perfilação da Expressão Gênica , Genoma Fúngico/genética , Podospora/crescimento & desenvolvimento , Podospora/genética , Fenômenos Biológicos/efeitos dos fármacos , Cobre/metabolismo , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos/genética , Ferro/metabolismo , Podospora/citologia , Podospora/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sideróforos/metabolismoRESUMO
The PaAlr1 gene encoding a putative plasma membrane magnesium (Mg) transporter in Podospora anserina was inactivated. The PaAlr1(Δ) mutants showed sensitivity to deprivation and excess Mg(2+) and Ca(2+). They also exhibited an autonomous ascospore maturation defect. Mutant ascospores were arrested at an early stage when they contained two nuclei. These data emphasize the role of Mg ions during sexual development in a filamentous fungus.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Podospora/enzimologia , Podospora/crescimento & desenvolvimento , Esporos Fúngicos/enzimologia , Esporos Fúngicos/crescimento & desenvolvimento , Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Técnicas de Inativação de Genes , Podospora/citologia , Podospora/metabolismo , Esporos Fúngicos/citologia , Esporos Fúngicos/metabolismoRESUMO
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/genéticaRESUMO
Prion diseases are associated with accumulation of the amyloid form of the prion protein, but the mechanisms of toxicity are unknown. Amyloid toxicity is also associated with fungal prions. In Podospora anserina, the simultaneous presence of [Het-s] prion and its allelic protein HET-S causes cell death in a self-/nonself-discrimination process. Here, using the prion form of a fragment of HET-s ([PrD(157)(+)]), we show that [Het-s]-HET-S toxicity can be faithfully recapitulated in yeast. Overexpression of Hsp40 chaperone, Sis1, rescues this toxicity by curing cells of [PrD(157)(+)]. We find no evidence for toxic [PrD(157)(+)] conformers in the presence of HET-S. Instead, [PrD(157)(+)] appears to seed HET-S to accumulate at the cell periphery and to form aggregates distinct from visible [PrD(157)(+)] aggregates. Furthermore, HET-S mutants that cause HET-S to be sequestered into [PrD(157)(+)] prion aggregates are not toxic. The localization of HET-S at the cell periphery and its association with cell death was also observed in the native host Podospora anserina. Thus, upon interaction with [Het-s], HET-S localizes to the cell periphery, and this relocalization, rather than the formation of mixed HET-s/HET-S aggregates, is associated with toxicity.
Assuntos
Amiloide/metabolismo , Proteínas Fúngicas/análise , Proteínas Fúngicas/metabolismo , Podospora/citologia , Príons/metabolismo , Amiloide/análise , Amiloide/genética , Proteínas Fúngicas/genética , Expressão Gênica , Proteínas de Choque Térmico HSP40/genética , Mutação , Podospora/genética , Podospora/metabolismo , Príons/análise , Príons/genética , Multimerização Proteica , Estrutura Terciária de Proteína , Regulação para CimaRESUMO
Peroxisome biogenesis relies on two known peroxisome matrix protein import pathways that are mediated by the receptors PEX5 and PEX7. These pathways converge at the importomer, a peroxisome-membrane complex that is required for protein translocation into peroxisomes and consists of docking and RING-finger subcomplexes. In the fungus Podospora anserina, the RING-finger peroxins are crucial for meiocyte formation, while PEX5, PEX7 or the docking peroxin PEX14 are not. Here we show that PEX14 and the PEX14-related protein PEX14/17 are differentially involved in peroxisome import during development. PEX14/17 activity does not compensate for loss of PEX14 function, and elimination of both proteins has no effect on meiocyte differentiation. In contrast, the docking peroxin PEX13, and the peroxins implicated in peroxisome membrane biogenesis PEX3 and PEX19, are required for meiocyte formation. Remarkably, the PTS2 coreceptor PEX20 is also essential for meiocyte differentiation and this function does not require PEX5 or PEX7. This finding suggests that PEX20 can mediate the import receptor activity of specific peroxisome matrix proteins. Our results suggest a new pathway for peroxisome import, which relies on PEX20 as import receptor and which seems critically required for specific developmental processes, like meiocyte differentiation in P. anserina.
Assuntos
Proteínas Fúngicas/metabolismo , Meiose , Peroxissomos/metabolismo , Podospora/citologia , Podospora/metabolismo , Proteínas Fúngicas/genética , Família Multigênica , Peroxissomos/genética , Podospora/genética , Transporte ProteicoRESUMO
Aging of biological systems is a fundamental process controlled by a complex network of molecular pathways. In the filamentous fungus Podospora anserina, a model in which organismal aging can conveniently be analysed, mitochondria play a central role. A wide range of relevant pathways were identified that contribute to the maintenance of a population of functional mitochondria. These pathways act in a hierarchical manner, but all the pathways are limited in capacity. At the end of the life cycle, when the various surveillance pathways are overwhelmed and damage has passed certain thresholds, programmed cell death brings the life of individual P. anserina to an end.
Assuntos
Envelhecimento/fisiologia , Expectativa de Vida , Mitocôndrias/fisiologia , Podospora/fisiologia , Morte Celular , Sequestradores de Radicais Livres/metabolismo , Humanos , Redes e Vias Metabólicas , Estresse Oxidativo , Podospora/citologia , Espécies Reativas de Oxigênio/metabolismoAssuntos
Adaptação Fisiológica , Aerobiose/genética , Carotenoides/biossíntese , Técnicas de Cultura de Células , Micélio/genética , Podospora/genética , Cromatografia Líquida de Alta Pressão , Contagem de Colônia Microbiana , Cruzamentos Genéticos , Variação Genética , Instabilidade Genômica , Melaninas/biossíntese , Viabilidade Microbiana , Microscopia , Micélio/citologia , Micélio/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Podospora/citologia , Podospora/metabolismo , Seleção GenéticaRESUMO
Cyclophilin D (CYPD) is a mitochondrial peptidyl prolyl-cis,trans-isomerase involved in opening of the mitochondrial permeability transition pore (mPTP). CYPD abundance increases during aging in mammalian tissues and in the aging model organism Podospora anserina. Here, we show that treatment of the P. anserina wild-type with low concentrations of the cyclophilin inhibitor cyclosporin A (CSA) extends lifespan. Transgenic strains overexpressing PaCypD are characterized by reduced stress tolerance, suffer from pronounced mitochondrial dysfunction and are characterized by accelerated aging and induction of cell death. Treatment with CSA leads to correction of mitochondrial function and lifespan to that of the wild-type. In contrast, PaCypD deletion strains are not affected by CSA within the investigated concentration range and show increased resistance against inducers of oxidative stress and cell death. Our data provide a mechanistic link between programmed cell death (PCD) and organismal aging and bear implications for the potential use of CSA to intervene into biologic aging.
Assuntos
Apoptose , Ciclofilinas/metabolismo , Podospora/citologia , Podospora/crescimento & desenvolvimento , Apoptose/efeitos dos fármacos , Peptidil-Prolil Isomerase F , Ciclofilinas/antagonistas & inibidores , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Genes involved in non-self recognition and host defence are typically capable of rapid diversification and exploit specialized genetic mechanism to that end. Fungi display a non-self recognition phenomenon termed heterokaryon incompatibility that operates when cells of unlike genotype fuse and leads to the cell death of the fusion cell. In the fungus Podospora anserina, three genes controlling this allorecognition process het-d, het-e and het-r are paralogs belonging to the same hnwd gene family. HNWD proteins are STAND proteins (signal transduction NTPase with multiple domains) that display a WD-repeat domain controlling recognition specificity. Based on genomic sequence analysis of different P. anserina isolates, it was established that repeat regions of all members of the gene family are extremely polymorphic and undergoing concerted evolution arguing for frequent recombination within and between family members. RESULTS: Herein, we directly analyzed the genetic instability and diversification of this allorecognition gene family. We have constituted a collection of 143 spontaneous mutants of the het-R (HNWD2) and het-E (hnwd5) genes with altered recognition specificities. The vast majority of the mutants present rearrangements in the repeat arrays with deletions, duplications and other modifications as well as creation of novel repeat unit variants. CONCLUSIONS: We investigate the extreme genetic instability of these genes and provide a direct illustration of the diversification strategy of this eukaryotic allorecognition gene family.
Assuntos
Proteínas Fúngicas/genética , Podospora/genética , Proteínas Fúngicas/química , Mutação , Podospora/citologia , Podospora/fisiologia , Estrutura Terciária de ProteínaRESUMO
In the filamentous fungus Podospora anserina, aging is systematically associated with mitochondrial DNA (mtDNA) instability. A causal link between deficiency of the cytochrome respiratory pathway and lifespan extension has been demonstrated. Knock out of the cytochrome respiratory pathway induces the expression of an alternative oxidase and is associated with a reduction in free radical production. The question of the links between mtDNA stability, ROS generation and lifespan is therefore clearly raised in this organism. NADPH lies at the heart of many anti-oxidant defenses of the cell. In Saccharomyces cerevisiae, the mitochondrial NADPH is largely provided by the Pos5 NADH kinase. We show here that disruption of PaNdk1 encoding the potential mitochondrial NADH kinase of P. anserina leads to severe somatic and sexual defects and to hypersensitivity to hydrogen peroxide and paraquat. Surprisingly, it also leads to a spectacular increase of mtDNA stability and lifespan. We propose that an adaptative metabolic change including the induction of the alternative oxidase can account for these results.
Assuntos
DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Podospora/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/genética , DNA Mitocondrial/genética , Deleção de Genes , Genes Fúngicos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , NAD/metabolismo , Estresse Oxidativo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Podospora/citologia , Podospora/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Superóxidos/metabolismoRESUMO
In previous investigations an impact of cellular copper homeostasis on ageing of the ascomycete Podospora anserina has been demonstrated. Here we provide new data indicating that mitochondria play a major role in this process. Determination of copper in the cytosolic fraction using total reflection X-ray fluorescence spectroscopy analysis and eGfp reporter gene studies indicate an age-related increase of cytosolic copper levels. We show that components of the mitochondrial matrix (i.e. eGFP targeted to mitochondria) become released from the organelle during ageing. Decreasing the accessibility of mitochondrial copper in P. anserina via targeting a copper metallothionein to the mitochondrial matrix was found to result in a switch from a copper-dependent cytochrome-c oxidase to a copper-independent alternative oxidase type of respiration and results in lifespan extension. In addition, we demonstrate that increased copper concentrations in the culture medium lead to the appearance of senescence biomarkers in human diploid fibroblasts (HDFs). Significantly, expression of copper-regulated genes is induced during in vitro ageing in medium devoid of excess copper suggesting that cytosolic copper levels also increase during senescence of HDFs. These data suggest that the identified molecular pathway of age-dependent copper dynamics may not be restricted to P. anserina but may be conserved from lower eukaryotes to humans.
Assuntos
Envelhecimento/fisiologia , Senescência Celular/fisiologia , Cobre/metabolismo , Fibroblastos/metabolismo , Podospora/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Fibroblastos/citologia , Regulação da Expressão Gênica , Humanos , Longevidade , Metalotioneína/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Podospora/citologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
ATP-binding cassette transporters are ubiquitous proteins that facilitate transport of diverse substances across a membrane. However, their exact role remains poorly understood. In order to test their function in a fungus life cycle, we deleted the two Podospora anserina peroxisomal ABC transporter pABC1 and pABC2 genes as well as the three genes involved in peroxisomal (fox2) and mitochondrial (scdA and echA) beta-oxidation. Analysis of the single and double mutants shows that fatty acid beta-oxidation occurs in both organelles. Furthermore, the peroxisomal and mitochondrial fatty acid beta-oxidation pathways are both dispensable for vegetative and sexual development. They are, however, differently required for ascospore pigmentation and germination, this latter defect being restored in a DeltapABC1 and DeltapABC2 background. We report also that lack of peroxisomal ABC transporters does not prevent peroxisomal long-chain fatty acid oxidation, suggesting the existence of another pathway for their import into peroxisomes. Finally, we show that some aspects of fatty acid degradation are clearly fungus species specific.