Calcium-induced release of cytochrome c from cardiolipin nanodisks: Implications for apoptosis.

Miniature bilayer membranes comprised of phospholipid and an apolipoprotein scaffold, termed nanodisks (ND), have been used in binding studies. When ND formulated with cardiolipin (CL), but not phosphatidylcholine, were incubated with cytochrome c, FPLC gel filtration chromatography provided evidence of a stable binding interaction. Incubation of CL ND with CaCl2 resulted in a concentration-dependent increase in sample turbidity caused by ND particle disruption. Prior incubation of CL ND with cytochrome c increased CL ND sensitivity to CaCl2-induced effects. Centrifugation of CaCl2-treated CL ND samples yielded pellet and supernatant fractions. Whereas the ND scaffold protein, apolipophorin III, was recovered in the pellet fraction along with CL, the majority of the cytochrome c pool was in the supernatant fraction. Moreover, when cytochrome c CL ND were incubated with CaCl2 at concentrations below the threshold to induce ND particle disruption, FPLC analysis showed that cytochrome c was released. Pre-incubation of CL ND with CaCl2 under conditions that do not disrupt ND particle integrity prevented cytochrome c binding to CL ND. Thus, competition between Ca2+ and cytochrome c for a common binding site on CL modulates cytochrome c binding and likely plays a role in its dissociation from CL-rich cristae membranes in response to apoptotic stimuli.