Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of contact sites

Percoll-purified rat liver mitochondria were shown to contain BAX dimer and rapidly (<2 min) release 5-10% of their cytochrome c when incubated in a s tandard KCl incubation medium under energized conditions. This release was not accompanied by release of adenylate kinase (AK), another intermembrane protein, and was not inhibited by Mg2+, dATP, inhibitors of the permeabilit y transition or ligands of the peripheral benzodiazepine receptor. However, release was greatly reduced by the presence of 5% (w/v) dextran (40 kDa), which caused a decrease in the light scattering (A(520)) of mitochondrial s uspensions. Dextran also inhibited both mitochondrial oxidation of exogenou s ferrocytochrome c in the presence of rotenone and antimycin, and respirat ory-chain-driven reduction of exogenous ferricytochrome c. Hypo-osmotic med ium or digitonin treatment of mitochondria caused a large additional releas e of both cytochrome c and AK that was not blocked by dextran. Polyaspartat e, which stabilizes the low conductance state of the voltage-dependent anio n channel (VDAC), increased cytochrome c release. VDAC and BAX are both fou nd at the contact sites between the inner and outer membranes and dextran i s known to stabilize these contact sites in isolated mitochondria. Thus our data suggest that regulation of a specific permeability pathway for cytoch rome c may be mediated by changes in protein-protein interactions within co ntact sites. The adenine nucleotide translocase is known to bind to VDAC an d thus provides an additional link between the specific cytochrome c releas e pathway and the permeability transition.