Oligomeric C-terminal truncated Bax preferentially releases cytochrome c but not adenylate kinase from mitochondria, outer membrane vesicles and proteoliposomes

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 (Bax DeltaC). Micromolar concentrations of Bax DeltaC released cytochrome c from isolated rat heart and liver mitochondria, while the release of adenylate kinase was not significantly affected. Bax DeltaC also released cytochrome c but not adenylate kinase from outer membrane vesicles filled with these proteins. However, Bax DeltaC 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. Bax DeltaC 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, Bax DeltaC 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 Bax DeltaC. Bax DeltaC 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) Bax DeltaC 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 Bax DeltaC action. (3) A limited pool of cytochrome c becomes the first target for Bax DeltaC. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.