Alexandra Werner

Supramolecular Organization of Cytochrome c Oxidase- and Alternative Oxidase-dependent Respiratory Chains in the Filamentous Fungus Podospora anserina

To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian “respirasome” model (Schägger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777–1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I2 and I2III2), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase- or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.

Copper-Modulated Gene Expression and Senescence in the Filamentous Fungus Podospora anserina

ABSTRACT We have previously shown that the control of cellular copper homeostasis by the copper-modulated transcription factor GRISEA has an important impact on the phenotype and lifespan of Podospora anserina. Here we demonstrate that copper depletion leads to the induction of an alternative respiratory pathway and to an increase in lifespan. This response compensates mitochondrial dysfunctions via the expression of PaAox, a nuclear gene coding for an alternative oxidase. It resembles the retrograde response inSaccharomyces cerevisiae. In P. anserina, this pathway appears to be induced by specific impairments of the copper-dependent cytochrome c oxidase. It is not induced as the result of a general decline of mitochondrial functions during senescence. We cloned and characterized PaAox. Transcript levels are decreased when cellular copper, superoxide, and hydrogen peroxide levels are raised. Copper also controls transcript levels ofPaSod2, the gene encoding the mitochondrial manganese superoxide dismutase (PaSOD2). PaSod2 is a target of transcription factor GRISEA. During the senescence of wild-type strain s, the activity of PaSOD2 decreases, whereas the activity of the cytoplasmic copper/zinc superoxide dismutase (PaSOD1) increases. Collectively, the data explain the postponed senescence of mutant grisea as a defined consequence of copper depletion, ultimately leading to a reduction of oxidative stress. Moreover, they suggest that during senescence of the wild-type strain, copper is released from mitochondria. The involved mechanism is unknown. However, it is striking that the permeability of mitochondrial membranes in animal systems changes during apoptosis and that mitochondrial proteins with an important impact on this type of cellular death are released.

Respiration, copper availability and SOD activity in P. anserina strains with different lifespan.

P. anserina mutants with impairments in complex IV (COX) of the respiratory chain are characterized by an increase in lifespan. Examples are the nuclear grisea mutant with a moderate lifespan extension (60%) and the immortal extranuclear ex1 mutant. Here we report data demonstrating that in mutant ex1 the level of the alternative oxidase (PaAOX) is significantly higher than in mutant grisea. PaAOX levels appear to be reversely dependent on COX activity. The activity profile of superoxide dismutases in the ex1 mutant resembles the profile in senescent wild-type cultures with a high cytoplasmic copper/zinc superoxide dismutase (PaSOD1) and a low mitochondrial manganese superoxide dismutase (PaSOD2) activity. In the grisea mutant, PaSOD1 activity is only detectable in cultures grown in copper-supplemented medium. The two copper-regulated genes PaCtr3 (coding for a high affinity copper transporter) and PaSod2 are not expressed in the two mutants grown in standard medium. The repression of these genes as well as the activity of PaSOD1 is dependent on the availability of cellular copper, which appears to be high in COX-deficient strains such as mutant ex1 and in the senescent wild-type strain. In the wild-type, changes in the cellular localization of copper and in the delivery of this metal to different proteins appear to occur during senescence. Collectively, the data explain the characteristic lifespan of the investigated strains as the result of differences in energy transduction and in the machinery protecting against oxidative stress.

Reactive oxygen species target specific tryptophan site in the mitochondrial ATP synthase.

The release of reactive oxygen species (ROS) as side products of aerobic metabolism in the mitochondria is an unavoidable consequence. As the capacity of organisms to deal with this exposure declines with age, accumulation of molecular damage caused by ROS has been defined as one of the central events during the ageing process in biological systems as well as in numerous diseases such as Alzheimers and Parkinsons Dementia. In the filamentous fungus Podospora anserina, an ageing model with a clear defined mitochondrial etiology of ageing, in addition to the mitochondrial aconitase the ATP synthase alpha subunit was defined recently as a hot spot for oxidative modifications induced by ROS. In this report we show, that this reactivity is not randomly distributed over the ATP Synthase, but is channeled to a single tryptophan residue 503. This residue serves as an intra-molecular quencher for oxidative species and might also be involved in the metabolic perception of oxidative stress or regulation of enzyme activity. A putative metal binding site in the proximity of this tryptophan residue appears to be crucial for the molecular mechanism for the selective targeting of oxidative damage.

A Genome-Wide Longitudinal Transcriptome Analysis of the Aging Model Podospora anserine

Aging of biological systems is controlled by various processes which have a potential impact on gene expression. Here we report a genome-wide transcriptome analysis of the fungal aging model Podospora anserina. Total RNA of three individuals of defined age were pooled and analyzed by SuperSAGE (serial analysis of gene expression). A bioinformatics analysis identified different molecular pathways to be affected during aging. While the abundance of transcripts linked to ribosomes and to the proteasome quality control system were found to decrease during aging, those associated with autophagy increase, suggesting that autophagy may act as a compensatory quality control pathway. Transcript profiles associated with the energy metabolism including mitochondrial functions were identified to fluctuate during aging. Comparison of wild-type transcripts, which are continuously down-regulated during aging, with those down-regulated in the long-lived, copper-uptake mutant grisea, validated the relevance of age-related changes in cellular copper metabolism. Overall, we (i) present a unique age-related data set of a longitudinal study of the experimental aging model P. anserina which represents a reference resource for future investigations in a variety of organisms, (ii) suggest autophagy to be a key quality control pathway that becomes active once other pathways fail, and (iii) present testable predictions for subsequent experimental investigations.

Alternative Oxidase Dependent Respiration Leads to an Increased Mitochondrial Content in Two Long-Lived Mutants of the Ageing Model Podospora anserina

The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms.

OXPHOS Supercomplexes Respiration and Life-Span Control in the Aging Model Podospora anserina

Abstract:  Recent biochemical evidence has indicated the existence of respiratory supercomplexes as well as ATP synthase oligomers in the inner mitochondrial membrane of different eukaryotes. We have studied the organization of the respiratory chain of a wild‐type strain and of two long‐lived mutants of the filamentous fungus Podospora anserina. This aging model is able to respire by either the standard or the alternative pathway. In the latter, electrons are directly transferred from ubiquinol to the alternative oxidase (AOX) and thus bypass complexes III and IV. We showed that the two pathways are composed of distinct respiratory supercomplexes. These data are of significance for the understanding of both respiratory pathways as well as of life‐span control and aging.

Genome analysis of filamentous fungi : identification of a highly conserved simple repetitive sequence in different strains of Podospora anserina

Abstract A nucleotide stretch located upstream of the MRAS3 gene of the zygomycete Mucor racemosus was found to hybridize to multiple restriction fragments of genomic DNA of the ascomycete Podospora anserina . DNA of strains derived from different geographical locations revealed no major restriction length fragment polymorphisms. Also, no age-related polymorphisms were observed. The repetitive sequence is located on all of the physically fractionated Podospora chromosomes. Sequence analysis of the upstream sequence of MRAS3 and of a DNA-stretch from a genomic clone of P. anserina which hybridized to the Mucor probe revealed a region of sequence homology. This sequence is composed of alternating G and T residues [poly(dG-dT) or poly(GT)] of 76 bp in length and thus represents a simple repetitive sequence, or microsatellite. A synthetic oligonucleotide, (GT) 8 , corresponding to this sequence gave also rise to a complex hybridization pattern. Screening of genomic libraries of P. anserina revealed poly(GT)-stretches on about 5–7% of all clones. Finally, this repetitive sequence was identified in the genome of the filamentous fungi Sordaria macrospora and Curvularia lunata .

A potential impact of DNA repair on ageing and lifespan in the ageing model organism Podospora anserina: Decrease in mitochondrial DNA repair activity during ageing

The free radical theory of ageing states that ROS play a key role in age-related decrease in mitochondrial function via the damage of mitochondrial DNA (mtDNA), proteins and lipids. In the sexually reproducing ascomycete Podospora anserina ageing is, as in other eukaryotes, associated with mtDNA instability and mitochondrial dysfunction. Part of the mtDNA instabilities may arise due to accumulation of ROS induced mtDNA lesions, which, as previously suggested for mammals, may be caused by an age-related decrease in base excision repair (BER). Alignments of known BER protein sequences with the P. anserina genome revealed high homology. We report for the first time the presence of BER activities in P. anserina mitochondrial extracts. DNA glycosylase activities decrease with age, suggesting that the increased mtDNA instability with age may be caused by decreased ability to repair mtDNA damage and hence contribute to ageing and lifespan control in this ageing model. Additionally, we find low DNA glycosylase activities in the long-lived mutants grisea and DeltaPaCox17::ble, which are characterized by low mitochondrial ROS generation. Overall, our data identify a potential role of mtDNA repair in controlling ageing and life span in P. anserina, a mechanism possibly regulated in response to ROS levels.

Age-related changes in the mitochondrial proteome of the fungus Podospora anserina analyzed by 2D-DIGE and LC–MS/MS

UNLABELLED Many questions concerning the molecular processes during biological aging remain unanswered. Since mitochondria are central players in aging, we applied quantitative two-dimensional difference gel electrophoresis (2D-DIGE) coupled to protein identification by mass spectrometry to study the age-dependent changes in the mitochondrial proteome of the fungus Podospora anserina - a well-established aging model. 67 gel spots exhibited significant, but remarkably moderate intensity changes. While typically the observed changes in protein abundance occurred progressively with age, for several proteins a pronounced change was observed at late age, sometimes inverting the trend observed at younger age. The identified proteins were assigned to a wide range of metabolic pathways including several implicated previously in biological aging. An overall decrease for subunits of complexes I and V of oxidative phosphorylation was confirmed by Western blot analysis and blue-native electrophoresis. Changes in several groups of proteins suggested a general increase in protein biosynthesis possibly reflecting a compensatory mechanism for increased quality control-related protein degradation at later age. Age-related augmentation in abundance of proteins involved in biosynthesis, folding, and protein degradation pathways sustain these observations. Furthermore, a significant decrease of two enzymes involved in the degradation of γ-aminobutyrate (GABA) supported its previously suggested involvement in biological aging. BIOLOGICAL SIGNIFICANCE We have followed the time course of changes in protein abundance during aging of the fungus P. anserina. The observed moderate but significant changes provide insight into the molecular adaptations to biological aging and highlight the metabolic pathways involved, thereby offering new leads for future research.