Omics-based approaches for the systematic profiling of mitochondrial biology.

Mitochondria are essential for cellular functions such as metabolism and apoptosis. They dynamically adapt to the changing environmental demands by adjusting their protein, nucleic acid, metabolite, and lipid contents. In addition, the mitochondrial components are modulated on different levels in response to changes, including abundance, activity, and interaction. A wide range of omics-based approaches has been developed to be able to explore mitochondrial adaptation and how mitochondrial function is compromised in disease contexts. Here, we provide an overview of the omics methods that allow us to systematically investigate the different aspects of mitochondrial biology. In addition, we show examples of how these methods have provided new biological insights. The emerging use of these toolboxes provides a more comprehensive understanding of the processes underlying mitochondrial function.

Global mitochondrial protein import proteomics reveal distinct regulation by translation and translocation machinery.

Most mitochondrial proteins are translated in the cytosol and imported into mitochondria. Mutations in the mitochondrial protein import machinery cause human pathologies. However, a lack of suitable tools to measure protein uptake across the mitochondrial proteome has prevented the identification of specific proteins affected by import perturbation. Here, we introduce mePRODmt, a pulsed-SILAC based proteomics approach that includes a booster signal to increase the sensitivity for mitochondrial proteins selectively, enabling global dynamic analysis of endogenous mitochondrial protein uptake in cells. We applied mePRODmt to determine protein uptake kinetics and examined how inhibitors of mitochondrial import machineries affect protein uptake. Monitoring changes in translation and uptake upon mitochondrial membrane depolarization revealed that protein uptake was extensively modulated by the import and translation machineries via activation of the integrated stress response. Strikingly, uptake changes were not uniform, with subsets of proteins being unaffected or decreased due to changes in translation or import capacity.