Examination of cytochrome c release from mitochondria during apoptosis

Mitochondria have assumed a central role in the mediation of apoptosis. They sequester apoptotic factors such as cytochrome C (CytC) within the mitochondria! inter-membrane space. The release of these factors into the cytosol consecrates an irreversible commitment to apoptotic death via caspase activation. Extensive research aimed at elucidating the mechanisms and components responsible for transport of these CytC across the outer-mitochondrial membrane has produced several competing theories. Importantly, a direct interaction between CytC and alleged protein transport complexes during release has never been demonstrated. To this end, recombinant molecules encoding specially selected, conditional globular domains at the C-terminus of human CytC have been generated, cloned, and expressed in vitro. Experiments were conducted to incorporate these recombinant CytC fusion proteins into isolated mitochondria from rodent liver tissue. Despite numerous reports indicating that CytC is imported by such mitochondrial preparations with a high efficiency, this was not observed here. Extensive experimentation to import these recombinant CytC molecules into isolated mitochondria was unsuccessful, precluding the use of this system to assess the apoptotic response of these proteins. The recombinant CytC sequences were then cloned into mammalian expression vector to be tested in cell culture models of apoptosis. Mouse embryonic stem cells null for CytC (CytC -/-) (ATCC) were selected as the best cell culture model to test the apoptotic behavior of the recombinant CytC molecules. However, attempts to express the recombinant CytC molecules in the mitochondria of these cells have not been successful thus far partly because the cells were frequently corrupted beyond usefulness.