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Cytochrome c causes pore formation in cardiolipin-containing membranes.

Proceedings of the National Academy of Sciences of the United States of America (2013-04-12)
Chris L Bergstrom, Paul A Beales, Yang Lv, T Kyle Vanderlick, John T Groves
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cytochrome c from Saccharomyces cerevisiae, ≥85% based on Mol. Wt. 12,588 basis
Sigma-Aldrich
ProteoMass Cytochrome c MALDI-MS Standard, vial of 10 nmol, (M+H+) 12,361.96 Da by calculation
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Cytochrome c from equine heart, BioUltra, ≥99% (SDS-PAGE), powder, suitable for mammalian cell culture
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Cytochrome c from pigeon breast muscle, ≥95% based on Mol. Wt. 12,173 basis
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Cytochrome c from bovine heart, ≥95% based on Mol. Wt. 12,327 basis
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Cytochrome c from bovine heart, ≥95% based on Mol. Wt. 12,327 basis, powder, suitable for mammalian cell culture
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Cytochrome c from equine heart, BioReagent, suitable for GFC marker
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Cytochrome c from equine heart, ≥95% (SDS-PAGE)
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Cytochrome c from equine heart, ≥95% based on Mol. Wt. 12,384 basis