The Mechanisms of the Release of Cytochrome c from Mitochondria Revealed by Proteomics Analysis

Abstract

Mitochondrial permeability transition (PT) is the phenomenon in which the mitochondrial inner membrane becomes permeable to various solutes and ions. When PT is induced by Ca(2+), cytochrome c is released from mitochondria into the cytosol where it then triggers subsequent steps of programmed cell death, apoptosis. Thus, the proteins that regulate PT and cytochrome c release could become druggable targets for various diseases. However, the mechanisms of PT and the release of cytochrome c have not yet been revealed. We previously showed that valinomycin, a potassium selective ionophore, also caused release of cytochrome c from mitochondria without inducing PT. This result indicates that cytochrome c could be released from mitochondria with or without induction of PT. In this study, to understand the difference of effects of valinomycin and Ca(2+) on mitochondria, we examined what protein species are released from valinomycin- and Ca(2+)-treated mitochondria by LC-MS/MS. As a result, only the proteins located in the intermembrane space were found to be released from valinomycin-treated mitochondria, while those in both the intermembrane space and in the matrix were released from Ca(2+)-treated mitochondria. Furthermore, the protein releases by each reagent occurred not selectively but in a concentration-dependent manner. Based on these results, the permeabilization effects of Ca(2+) and valinomycin on the inner and outer mitochondrial membranes are discussed.