Identification of proteins differentially regulated upon perturbed mitochondrial gene expression

The aim of this project was to search for the new factors engaged in mitochondrial nucleic acids metabolism under stress conditions. To this end we treated human 293 cells with mitochondrial replication/transcription inhibitor ethidium bromide and we compared proteomes of untreated vs treated cells. We were looking for the proteins whose level would be differentially regulated in response to disturbed mitochondrial gene expression. In our studies we utilized a stable 293 cell line that inducible expresses a fusion of EGFP with mitochondria targeting sequence. This approach enabled us to control mitochondrial import by analysis of the abundance of mitochondrial EGFP marker. We isolated highly purified mitochondria from the cells, lysed them and subjected protein extracts to labelling with iTRAQ reagents. We performed quantitative analysis of stress- induced changes of human mitochondrial proteome with the use of MaxQuant software.