Mutations in the phosphatase domain of the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase result in the transcriptional activation of the alternative oxidase and gluconeogenic pathways in Podospora anserina.

By screening suppressors of a respiratory mutant lacking a functional cytochrome pathway in the filamentous fungus Podospora anserina, we isolated a mutation located in the phosphatase domain of the bi-functional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2). We show that the inactivation of the phosphatase but not of the kinase domain is responsible for the suppressor effect that results from the activation of the RSEs transcription factors that control expression of AOX, an alternative oxidase able to bypass the mitochondria cytochrome pathway of respiration. Remarkably, activation of the RSEs also stimulates the expression of the gluconeogenic enzymes, fructose-1,6 bi-phosphatase (FBPase-1) and phosphoenolpyruvate carboxykinase (PCK-1). We thus reveal in P. anserina an apparently paradoxical situation where the inactivation of the phosphatase domain of PFK-2/FBPase-2, supposed to stimulate glycolysis, is correlated with the transcriptional induction of the gluconeogenic enzymes. Phylogenic analysis revealed the presence of multiple presumed PFK-2/FBPase-2 isoforms in all the species of tested Ascomycetes.

Regulation of Aerobic Energy Metabolism in Podospora anserina by Two Paralogous Genes Encoding Structurally Different c-Subunits of ATP Synthase.

Most of the ATP in living cells is produced by an F-type ATP synthase. This enzyme uses the energy of a transmembrane electrochemical proton gradient to synthesize ATP from ADP and inorganic phosphate. Proton movements across the membrane domain (FO) of the ATP synthase drive the rotation of a ring of 8-15 c-subunits, which induces conformational changes in the catalytic part (F1) of the enzyme that ultimately promote ATP synthesis. Two paralogous nuclear genes, called Atp9-5 and Atp9-7, encode structurally different c-subunits in the filamentous fungus Podospora anserina. We have in this study identified differences in the expression pattern for the two genes that correlate with the mitotic activity of cells in vegetative mycelia: Atp9-7 is transcriptionally active in non-proliferating (stationary) cells while Atp9-5 is expressed in the cells at the extremity (apex) of filaments that divide and are responsible for mycelium growth. When active, the Atp9-5 gene sustains a much higher rate of c-subunit synthesis than Atp9-7. We further show that the ATP9-7 and ATP9-5 proteins have antagonist effects on the longevity of P. anserina. Finally, we provide evidence that the ATP9-5 protein sustains a higher rate of mitochondrial ATP synthesis and yield in ATP molecules per electron transferred to oxygen than the c-subunit encoded by Atp9-7. These findings reveal that the c-subunit genes play a key role in the modulation of ATP synthase production and activity along the life cycle of P. anserina. Such a degree of sophistication for regulating aerobic energy metabolism has not been described before.

Deletion of the MED13 and CDK8 subunits of the Mediator improves the phenotype of a long-lived respiratory deficient mutant of Podospora anserina.

In Podospora anserina, the loss of function of the cytochrome segment of the mitochondrial respiratory chain is viable. This is due to the presence in this organism, as in most filamentous fungi, of an alternative respiratory oxidase (AOX) that provides a bypass to the cytochrome pathway. However mutants lacking a functional cytochrome pathway present multiple phenotypes including poorly colored thin mycelium and slow growth. In a large genetic screen based on the improvement of these phenotypes, we isolated a large number of independent suppressor mutations. Most of them led to the constitutive overexpression of the aox gene. In this study, we characterize a new suppressor mutation that does not affect the production of AOX. It is a loss-of-function mutation in the gene encoding the MED13 subunit of the kinase module of the Mediator complex. Inactivation of the cdk8 gene encoding another subunit of the same module also results in partial suppression of a cytochrome-deficient mutant. Analysis of strains lacking the MED13 or CDK8 subunits points to the importance of these subunits as regulators involved in diverse physiological processes such as growth, longevity and sexual development. Interestingly, transcriptional analyses indicate that in P. anserina, loss of the respiratory cytochrome pathway results in the up-regulation of glycolysis-related genes revealing a new type of retrograde regulation. The loss of MED13 augments the up-regulation of some of these genes.

Caenorhabditis elegans expressing the Saccharomyces cerevisiae NADH alternative dehydrogenase Ndi1p, as a tool to identify new genes involved in complex I related diseases.

Isolated complex I deficiencies are one of the most commonly observed biochemical features in patients suffering from mitochondrial disorders. In the majority of these clinical cases the molecular bases of the diseases remain unknown suggesting the involvement of unidentified factors that are critical for complex I function. The Saccharomyces cerevisiae NDI1 gene, encoding the mitochondrial internal NADH dehydrogenase was previously shown to complement a complex I deficient strain in Caenorhabditis elegans with notable improvements in reproduction and whole organism respiration. These features indicate that Ndi1p can functionally integrate the respiratory chain, allowing complex I deficiency complementation. Taking into account the Ndi1p ability to bypass complex I, we evaluate the possibility to extend the range of defects/mutations causing complex I deficiencies that can be alleviated by NDI1 expression. We report here that NDI1 expressing animals unexpectedly exhibit a slightly shortened lifespan, a reduction in the progeny, and a depletion of the mitochondrial genome. However, Ndi1p is expressed and targeted to the mitochondria as a functional protein that confers rotenone resistance to those animals without affecting their respiration rate and ATP content. We show that the severe embryonic lethality level caused by the RNAi knockdowns of complex I structural subunit encoding genes (e.g., NDUFV1, NDUFS1, NDUFS6, NDUFS8, or GRIM-19 human orthologs) in wild type animals is significantly reduced in the Ndi1p expressing worm. All together these results open up the perspective to identify new genes involved in complex I function, assembly, or regulation by screening an RNAi library of genes leading to embryonic lethality that should be rescued by NDI1 expression.

Does autophagy mediate age-dependent effect of dietary restriction responses in the filamentous fungus Podospora anserina?

Autophagy is a well-conserved catabolic process, involving the degradation of a cell's own components through the lysosomal/vacuolar machinery. Autophagy is typically induced by nutrient starvation and has a role in nutrient recycling, cellular differentiation, degradation and programmed cell death. Another common response in eukaryotes is the extension of lifespan through dietary restriction (DR). We studied a link between DR and autophagy in the filamentous fungus Podospora anserina, a multicellular model organism for ageing studies and mitochondrial deterioration. While both carbon and nitrogen restriction extends lifespan in P. anserina, the size of the effect varied with the amount and type of restricted nutrient. Natural genetic variation for the DR response exists. Whereas a switch to carbon restriction up to halfway through the lifetime resulted in extreme lifespan extension for wild-type P. anserina, all autophagy-deficient strains had a shorter time window in which ageing could be delayed by DR. Under nitrogen limitation, only PaAtg1 and PaAtg8 mediate the effect of lifespan extension; the other autophagy-deficient mutants PaPspA and PaUth1 had a similar response as wild-type. Our results thus show that the ageing process impinges on the DR response and that this at least in part involves the genetic regulation of autophagy.

Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina.

In Podospora anserina, the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase (aox) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase (pck) and fructose-1,6-biphosphatase (fbp). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3. Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3. It showed that in addition to aox, fbp, and pck, RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn2Cys6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools.

Experimental relocation of the mitochondrial ATP9 gene to the nucleus reveals forces underlying mitochondrial genome evolution.

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.

Biological roles of the Podospora anserina mitochondrial Lon protease and the importance of its N-domain.

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.

Two nuclear life cycle-regulated genes encode interchangeable subunits c of mitochondrial ATP synthase in Podospora anserina.

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.

Molecular gene therapy: overexpression of the alternative NADH dehydrogenase NDI1 restores overall physiology in a fungal model of respiratory complex I deficiency.

Defects in oxidative phosphorylation lie at the heart of a wide variety of degenerative disorders, cancer, and aging. Here, we show, using the fungal model Podospora anserina, that the overexpression of the native mitochondrial matrix-faced type II NADH dehydrogenase NDI1, paralogue of the human apoptosis inducing factor AIF1, can fully restore all physiological consequences of respiratory complex I deficiency. We disrupted the 19.3-kDa subunit of the complex I catalytic core, orthologue of the human PSST subunit, leading to a complete absence of the complex without affecting the assembly and/or stability of the rest of the respiratory chain. This disruption caused a several-fold life span extension at the expense of both male and female fertility. The effect was generally similar but markedly milder than that caused by defects in the complex III/IV-dependent pathway and not associated with a clear reduction in the steady-state level of mitochondrial reactive oxygen species. Whereas the native expression of NDI1 was sufficient to overcome lethality, only the artificial, constitutive overexpression of NDI1 could fully remedy this deficiency: The latter strikingly restored both life span and fertility to levels indistinguishable from wild type, thus demonstrating its unique potential in molecular gene therapy.

Calorie restriction causes healthy life span extension in the filamentous fungus Podospora anserina.

Although most fungi appear to be immortal, some show systemic senescence within a distinct time frame. Podospora anserina for example shows an irreversible growth arrest within weeks of culturing associated with a destabilization of the mitochondrial genome. Here, we show that calorie restriction (CR), a regimen of under-nutrition without malnutrition, increases not only life span but also forestalls the aging-related decline in fertility. Similar to respiratory chain deficiencies the life span extension is associated with lower levels of intracellular H(2)O(2) measurements and a stabilization of the mitochondrial genome. Unlike respiratory chain deficiencies, CR cultures have a wild-type-like OXPHOS machinery similar to that of well-fed cultures as shown by native electrophoresis of mitochondrial protein complexes. Together, these data indicate that life span extension via CR is fundamentally different from that via respiratory chain mutations: Whereas the latter can be seen as a pathology, the former promotes healthy life span extension and may be an adaptive response.

Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina.

In Podospora anserina, inactivation of the respiratory chain results in a spectacular life-span extension. This inactivation is accompanied by the induction of the alternative oxidase. Although the functional value of this response is evident, the mechanism behind it is far from understood. By screening suppressors able to reduce the life-span extension of cytochrome-deficient mutants, we identified mutations in two zinc-cluster proteins, RSE2 and RSE3, which are conserved in other ascomycetes. These mutations led to the overexpression of the genes encoding the alternative oxidase and the gluconeogenic enzymes, fructose-1, 6 biphosphatase, and pyruvate carboxykinase. Both RSE2 and RSE3 are required for the expression of these genes. We also show that, even in the absence of a respiratory deficiency, the wild-type RSE2 and RSE3 transcription factors are involved in life-span control and their inactivation retards aging. These data are discussed with respect to aging, the regulation of the alternative oxidase, and carbon metabolism.

The genome sequence of the model ascomycete fungus Podospora anserina.

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.

Gene deletion and allelic replacement in the filamentous fungus Podospora anserina.

Gene replacement via homologous recombination is a fundamental tool for the analysis of gene function. However, this event is rare in organisms like the filamentous fungus Podospora anserina. We show here that deletion of the PaKu70 gene is an efficient strategy for improving gene manipulation in this organism. By using the DeltaPaKu70 strain, it is now possible (1) to produce deletion mutants with an efficiency of 100%, (2) to achieve allelic exchange by introducing a mutated allele associated with a selection cassette at the locus, (3) to introduce a mutation in a gene without co-insertion of a selectable marker and without any modification of the target locus.

A mutation in the gene encoding cytochrome c1 leads to a decreased ROS content and to a long-lived phenotype in the filamentous fungus Podospora anserina.

We present here the properties of a complex III loss-of-function mutant of the filamentous fungus Podospora anserina. The mutation corresponds to a single substitution in the second intron of the gene cyc1 encoding cytochrome c(1), leading to a splicing defect. The cyc1-1 mutant is long-lived, exhibits a defect in ascospore pigmentation, has a reduced growth rate and a reduced ROS production associated with a stabilisation of its mitochondrial DNA. We also show that increased longevity is linked with morphologically modified mitochondria and an increased number of mitochondrial genomes. Overexpression of the alternative oxidase rescues all these phenotypes and restores aging. Interestingly, the absence of complex III in this mutant is not paralleled with a deficiency in complex I activity as reported in mammals although the respiratory chain of P. anserina has recently been demonstrated to be organized according to the "respirasome" model.

Identification and biochemical analysis of a mitochondrial endonuclease of Podospora anserina related to curved-DNA binding proteins.

We purified and characterized previously from Podospora anserina mitochondria an endonuclease, active on single-stranded, double-stranded and flap DNA, with RNAse H activity, named P49 according to the major 49 kDa band observed on SDS-PAGE. Edman sequencing allowed us to identify the corresponding gene called nuc49. Here we report the properties of the (His)-tagged NUC49 protein expressed in E. coli. We show that this protein does exhibit an endonuclease activity on plasmid DNA, circular recessed and flap M13 substrate with short protruding single strand. However, in contrast to the mt endonuclease purified fraction it does not present RNase H activity and does not cleave linear flap substrate. The activity differences between the protein expressed in E. coli and the mitochondrial endonuclease fraction previously described are discussed. NUC49 presents a strong homology with the S. pombe CDB4 curved DNA binding protein which belongs to a large family including the human cell cycle protein PA2G4 and is able to bind curved DNA. The results constitute the first description of a mitochondrial endonuclease activity associated to this family of proliferation associated homologous proteins. The function of this endonuclease either in recombination, repair or mt DNA rearrangements remains to be determined.

Interaction between the oxa1 and rmp1 genes modulates respiratory complex assembly and life span in Podospora anserina.

A causal link between deficiency of the cytochrome respiratory pathway and life span was previously shown in the filamentous fungus Podospora anserina. To gain more insight into the relationship between mitochondrial function and life span, we have constructed a strain carrying a thermosensitive mutation of the gene oxa1. OXA1 is a membrane protein conserved from bacteria to human. The mitochondrial OXA1 protein is involved in the assembly/insertion of several respiratory complexes. We show here that oxa1 is an essential gene in P. anserina. The oxa1(ts) mutant exhibits severe defects in the respiratory complexes I and IV, which are correlated with an increased life span, a strong induction of the alternative oxidase, and a reduction in ROS production. However, there is no causal link between alternative oxidase level and life span. We also show that in the oxa1(ts) mutant, the extent of the defects in complexes I and IV and the life-span increase depends on the essential gene rmp1. The RMP1 protein, whose function is still unknown, can be localized in the mitochondria and/or the cytosolic compartment, depending on the developmental stage. We propose that the RMP1 protein could be involved in the process of OXA1-dependent protein insertion.

Characterization of the genomic organization of the region bordering the centromere of chromosome V of Podospora anserina by direct sequencing.

A Podospora anserina BAC library of 4800 clones has been constructed in the vector pBHYG allowing direct selection in fungi. Screening of the BAC collection for centromeric sequences of chromosome V allowed the recovery of clones localized on either sides of the centromere, but no BAC clone was found to contain the centromere. Seven BAC clones containing 322,195 and 156,244bp from either sides of the centromeric region were sequenced and annotated. One 5S rRNA gene, 5 tRNA genes, and 163 putative coding sequences (CDS) were identified. Among these, only six CDS seem specific to P. anserina. The gene density in the centromeric region is approximately one gene every 2.8kb. Extrapolation of this gene density to the whole genome of P. anserina suggests that the genome contains about 11,000 genes. Synteny analyses between P. anserina and Neurospora crassa show that co-linearity extends at the most to a few genes, suggesting rapid genome rearrangements between these two species.

Dynamics of the mitochondrial genome during Podospora anserina aging.

Senescence in Podospora anserina is always correlated with extrachromosomal mitochondrial DNA amplification (senDNA). Here we report a quantitative kinetic analysis of the molecular events that occur in the mitochondrial DNA of several wild-type cultures during aging. For each culture, the amplification of senDNA molecules and the modifications of the mitochondrial chromosome are analyzed at different ages and in relation with two age-related parameters: growth rate and fertility. We find senDNAs exponentially amplified from the germination state and particular regions of the mitochondrial chromosome preferentially lost only in the pre-senescent state, i.e. a few centimeters before the growth arrest of the mycelium. This late loss of information, concomitant with the first phenotypic expression of senescence (loss of fertility), begins in the regions from which the senDNAs originate and culminates with the growth arrest of the mycelium.