Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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

ABSTRACT: Transcriptome profile of S. cerevisiae strains grown in rich ethanol/glycerol media, where the endogenous mitochondrial gene ATP9 has been deleted and relocated to the nucleus by expressing synthetic, codon-optimized versions of the Podospora anserina genes Atp9-5 and Atp9-7. The strains AMY7, AMY8, AMY10, and AMY11 each express one of these genes either from a centromeric or 2-micron plasmid. These strains are derived from RKY26, an atp9 deletion mutant which was not included in this experiment since it does not grow on respiratory media. The isogenic wild-type strain (MR6) was included as a control. Strains were produced for this study, and the arrays used were yeast tiling microarrays from Affymetrix. Note that normalization was performed using genomic DNA hybridizations, the raw data for which can be found in ArrayExpress Accession # E-TABM-1176.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Raeka Aiyar

PROVIDER: E-MTAB-1115 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The free radical theory of ageing is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Since the majority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the ageing model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidised and non-oxidised protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidised. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during ageing as long as protein de-novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged und undamaged protein species. These findings contradict a massive increase in protein oxidation during ageing and rather suggest a protein damage homeostasis mechanism even at late age.

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

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