The mechanism by which the proapoptotic protein Bax releases cytochrome c from mitochondria is not fully understood. The present work approaches this problem using C-terminal truncated oligomeric Bax (BaxΔC). Micromolar concentrations of BaxΔC released cytochrome c from isolated rat heart and liver mitochondria, while the release of adenylate kinase was not significantly affected. BaxΔC also released cytochrome c but not adenylate kinase from outer membrane vesicles filled with these proteins. However, BaxΔC was ineffective in releasing cytochrome c when outer membrane vesicles were obtained in the presence of glycerol, conditions under which the number of contact sites was drastically reduced. BaxΔC did not liberate encapsulated cytochrome c and adenylate kinase from pure phospholipid vesicles or vesicles reconstituted with porin. However, when the hexokinase–porin–adenine nucleotide translocase complex from brain mitochondria was reconstituted in vesicles, BaxΔC released internal cytochrome c but not adenylate kinase. In all these systems, only a small portion of total cytochrome c present in either mitochondria or vesicles could be liberated by BaxΔC. BaxΔC also increased the accessibility of external cytochrome c to either oxidation by complex IV or reduction by complex III in intact liver and heart mitochondria. Conclusions: (1) BaxΔC selectively releases cytochrome c and enables a bidirectional movement of cytochrome c across the outer mitochondrial membrane. (2) A multiprotein complex that resembles the mitochondrial contact sites is a prerequisite for BaxΔC action. (3) A limited pool of cytochrome c becomes the first target for BaxΔC.
