Mitochondria mediate key cellular processes, including energy production and cell death. Although mitochondrial proteases regulate these processes,^1,2 technology to study global proteolysis within mitochondria is underdeveloped. Our understanding of mitochondrial dynamics could be greatly improved with a proteomics technique that also identifies proteolytic events.

We have developed a new approach called ‘mito-TAILS’ that quantitatively profiles the mitochondrial proteome and proteolytic events by combining mitochondrial enrichment, SILAC,³ and a quantitative proteomics technique (terminal amine isotopic labeling of substrates; TAILS).⁴ 

In this study, mito-TAILS was used to characterize how the mitochondrial N-terminal proteome is affected by infection with enteropathogenic Escherichia coli (EPEC). EPEC virulence factors target mitochondria; they are imported, cleaved, and contribute to apoptosis of infected cells. Human epithelial cells were grown in light or heavy isotope-coded SILAC medium and either infected with wild-type EPEC or mock infected. Cells were harvested, pooled, and homogenized and mitochondria were isolated from 95% of the cell homogenate; 5% was used for a complementary whole cell analysis. N-terminal proteomes were quantitatively compared using TAILS and mass spectrometry.

We identified 1273 proteins and 2104 N-terminal peptides (1% FDR) from three replicates. Proteins from mitochondrial cellular compartments were significantly enriched in mitochondrial fractions (Gene Ontology). The ‘pathogenic E. coli infection’ pathway was enriched in whole cells (p=0.0136) and ‘oxidative phosphorylation’ was enriched in mitochondria (p=2.4E-6), indicating successful infection and mitochondrial enrichment. We identified changes in the abundance of mitochondrial import receptors and N-terminal peptides corresponding to increased removal of the mitochondrial transit peptide, suggesting altered import (e.g. mtHsp60, ATPase subunits). Finally, we identified known apoptotic events, including activating proteolysis of the mitochondrial protease HtrA2 during EPEC infection, which is then released to the cytoplasm, where we observe increased proteolysis of its cytoplasmic substrates from the whole cell sample at known HtrA2 cleavage sites.

Mito-TAILS is a valuable new approach to study mitochondrial proteome dynamics and proteolysis. To our knowledge, this is the first terminal proteomics study of human mitochondria. By profiling the cellular proteome alongside the mitochondrial proteome, this technique enables the investigation of cell-wide consequences of mitochondrial processes and can be applied to study other mitochondrial pathways and pathologies.