Cytochrome c causes pore formation in cardiolipin-containing membranes

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.

Oligopolyphenylenevinylene-Conjugated Oligoelectrolyte Membrane Insertion Molecules Selectively Disrupt Cell Envelopes of Gram-Positive Bacteria

“…Furthermore, the importance of DPG content in antimicrobial susceptibility has been demonstrated using model membrane systems (20,(42)(43)(44)(45). Additionally, apoptosis in mitochondria has been shown to be inextricably linked to DPG and its affinity for cytochrome c, with the DPG content increasing from Ϸ4% to in excess of 20% immediately prior to apoptosis (45)(46)(47). Therefore, the increased content of DPG in Gram-positive microbial membranes likely underlies the sensitivity of Gram-positive organisms to OPV-COEs.…”

“…Nisin interacts with the lipid precursor, lipid II, and can have up to a 700-fold increase in activity against active cells compared to protoplasts, an observation attributed to a general low-affinity permeability interaction with DPG and a targeted lipid II-dependent poration mechanism (46,47). It is unlikely that a similar lipid II-dependent mechanism exists with OPV-COEs, as it is an interaction driven by a specificity with the D-Ala-D-Ala pentapeptide chain that stabilizes the lipid II molecule, and no such specific binding site exists on OPV-COEs.…”

Role of Lysines in Cytochrome c–Cardiolipin Interaction

“…29 ■ EXPERIMENTAL PROCEDURES Construction of the Horse cyt c Expression System. A version of the horse cyt c synthetic gene was designed on the basis of the sequence of a previously reported cyt c synthetic gene 25,29 and its synthesis accomplished by Primm srl (Milan, Italy). The synthetic gene was flanked by the NcoI and BamHI restriction sites, at the 5′ and 3′ ends, respectively.…”

The key role played by charge in the interaction of cytochrome c with cardiolipin

“…In contrast to the previous investigation, where a positively charged residue was replaced by a polar side chain carrying no net charge (Lys → Asn mutation), in the present study the Lys residues have been replaced by Arg carrying a positive charge at neutral pH as Lys, but with a different structure, and the non-polar, aliphatic amino acid, Ala. The present study has been performed on ferric horse heart cyt c, which behaves similar to the human counterpart [29] and actively participates in cell apoptosis [4,[30][31][32][33].…”