Cytochrome <i>c</i> release from mitochondria proceeds by a two-step process

TLDR

Cytochrome c is often released from mitochondria during early apoptosis, but the precise mechanisms remain unclear. This study demonstrates that cytochrome c release requires a two‑step process. Cytochrome c exists in loosely and tightly bound pools to cardiolipin; first, cardiolipin interactions must be disrupted to solubilize the protein, then Bax‑mediated outer membrane permeabilization allows extrusion. Neither disrupting cardiolipin binding nor Bax permeabilization alone triggers release, and the two‑step mechanism is also impaired during mitochondrial permeability transition when cardiolipin interaction persists.

Abstract

Cytochrome c is often released from mitochondria during the early stages of apoptosis, although the precise mechanisms regulating this event remain unclear. In this study, with isolated liver mitochondria, we demonstrate that cytochrome c release requires a two-step process. Because cytochrome c is present as loosely and tightly bound pools attached to the inner membrane by its association with cardiolipin, this interaction must first be disrupted to generate a soluble pool of this protein. Specifically, solubilization of cytochrome c involves a breaching of the electrostatic and/or hydrophobic affiliations that this protein usually maintains with cardiolipin. Once cytochrome c is solubilized, permeabilization of the outer mitochondrial membrane by Bax is sufficient to allow the extrusion of this protein into the extramitochondrial environment. Neither disrupting the interaction of cytochrome c with cardiolipin, nor permeabilizing the outer membrane with Bax, alone, is sufficient to trigger this protein's release. This mechanism also extends to conditions of mitochondrial permeability transition insofar as cytochrome c release is significantly depressed when the electrostatic interaction between cytochrome c and cardiolipin remains intact. Our results indicate that the release of cytochrome c involves a distinct two-step process that is undermined when either step is compromised.