Cytochrome c causes pore formation in cardiolipin-containing membranes

The release of cytochrome c from mitochondria is a key signaling mechanism in apoptosis. Although extramitochondrial proteins are thought to initiate this release, the exact mechanisms remain unclear. Cytochrome c (cyt c ) binds to and penetrates lipid structures containing the inner mitochondrial membrane lipid cardiolipin (CL), leading to protein conformational changes and increased peroxidase activity. We describe here a direct visualization of a fluorescent cyt c crossing synthetic, CL-containing membranes in the absence of other proteins. We observed strong binding of cyt c to CL in phospholipid vesicles and bursts of cyt c leakage across the membrane. Passive fluorescent markers such as carboxyfluorescein and a 10-kDa dextran polymer crossed the membrane simultaneously with cyt c , although larger dextrans did not. The data show that these bursts result from the opening of lipid pores formed by the cyt c –CL conjugate. Pore formation and cyt c leakage were significantly reduced in the presence of ATP. We suggest a model, consistent with these findings, in which the formation of toroidal lipid pores is driven by initial cyt c -induced negative spontaneous membrane curvature and subsequent protein unfolding interactions. Our results suggest that the CL–cyt c interaction may be sufficient to allow cyt c permeation of mitochondrial membranes and that cyt c may contribute to its own escape from mitochondria during apoptosis.