title: Proteomic analysis of mitochondria from senescent Podospora anserina casts new light on ROS dependent aging mechanisms.
abstract: The mitochondrial free radical theory of aging (MFRTA) states that reactive oxygen species (ROS) generated at the respiratory electron transport chain are active in causing age-related damage of biomolecules like lipids, nucleic acids and proteins. Accumulation of this kind of damage results in functional impairments, aging and death of biological systems. Here we report data of an analysis to monitor the age-related quantitative protein composition of the mitochondria of the fungal aging model Podospora anserina. The impact of senescence on mitochondrial protein composition was analyzed by LC-MS. In an untargeted proteomic approach, we identified 795 proteins in samples from juvenile and senescent wild-type cultures and obtained quantitative information for 226 of these proteins by spectral counting. Despite the broad coverage of the proteome, no substantial changes in known age-related pathways could be observed. For a more detailed analysis, a targeted proteome analysis was applied focusing on 15 proteins from respiratory, ROS-scavenging and quality control pathways. Analyzing six distinct age-stages from juvenile to senescent P. anserina cultures revealed low, but statistically significant changes for the mitochondrial respiratory complexes. A P. anserina PaSod3 over-expression mutant with a phenotype of mitochondrial ROS over-production was used for biological evaluation of changes observed during aging. LC-MS analysis of the mutant revealed severe changes to the mitochondrial proteome--substantially larger than observed during senescence. Interestingly the amount of ATP synthase subunit g, involved in cristae formation is significantly decreased in the mutant implicating ROS-induced impairments in ATP synthase dimer and cristae formation. The difference between protein-profiles of aging wild type and ROS stressed mutant suggests that oxidative stress within the mitochondria is not the dominating mechanism for the aging process in P. anserina. Collectively, while our data do not exclude an effect of ROS on specific proteins and in signaling and control of pathways which are governing aging of P. anserina, it contradicts increasing ROS as a cause of a gross general and non-selective accumulation of damaged proteins during senescence. Instead, ROS may be effective by controlling specific regulators of mitochondrial function.
authors.affiliation: Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr University Bochum, D-44780 Bochum, Germany. Electronic address: Nicole.Plohnke@ruhr-uni-bochum.de.
authors.forename: Nicole
authors.initials: N
authors.lastname: Plohnke
authors.affiliation: Institute of Molecular Biosciences, Faculty for Biosciences and Cluster of Excellence Macromolecular Complexes, Johann Wolfgang Goethe University, D-60438 Frankfurt, Germany. Electronic address: a.hamann@bio.uni-frankfurt.de.
authors.forename: Andrea
authors.initials: A
authors.lastname: Hamann
authors.affiliation: Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr University Bochum, D-44780 Bochum, Germany. Electronic address: Ansgar.Poetsch@ruhr-uni-bochum.de.
authors.forename: Ansgar
authors.initials: A
authors.lastname: Poetsch
authors.affiliation: Institute of Molecular Biosciences, Faculty for Biosciences and Cluster of Excellence Macromolecular Complexes, Johann Wolfgang Goethe University, D-60438 Frankfurt, Germany. Electronic address: osiewacz@bio.uni-frankfurt.de.
authors.forename: Heinz D
authors.initials: HD
authors.lastname: Osiewacz
authors.affiliation: Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr University Bochum, D-44780 Bochum, Germany. Electronic address: Matthias.Roegner@ruhr-uni-bochum.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Rögner
authors.affiliation: Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr University Bochum, D-44780 Bochum, Germany. Electronic address: Sascha.Rexroth@ruhr-uni-bochum.de.
authors.forename: Sascha
authors.initials: S
authors.lastname: Rexroth
mesh_terms: D000375:Aging / Q000378:metabolism*; D056148:Chromatography, Reverse-Phase; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D018384:Oxidative Stress; D010641:Phenotype; D048468:Podospora / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry
chemical_list: D005656:Fungal Proteins; D024101:Mitochondrial Proteins; D017382:Reactive Oxygen Species
keywords: Free radical theory of aging; Mass spectrometry; Podospora anserina; Proteome analysis; Selected reaction monitoring