Mitochondria play key roles in apoptosis, a central step being the release of cytochrome c (cyt c) across the outer mitochondrial membrane into the cytoplasm. We review this process in terms of the influences that induce mitochondria to release cyt c, the possible mechanisms of such release and the downstream consequences for caspase activation. The contributions of members of the Bcl-2 family in regulating mitochondrial activities relevant to apoptotic signaling are considered. Antiapoptotic members, such as Bcl-2 itself, are antagonistic to other family members, which prominently include Bax amongst a host of other proapoptotic proteins homologous to Bcl-2. Focus is placed on technical methods of determining cyt c release, which encompass cell fractionation, biochemistry, immunochemistry and confocal microscopy [including observations of release in real time using cyt c-green fluorescent protein (GFP) fusion proteins]. The advantages and potential pitfalls of the various approaches are discussed, with some emphasis on the use of cyt c-GFP fusions and the determination of the characteristics of the putative outer membrane pore through which cyt c and other mitochondrial proteins with proapoptotic functions may pass. The richness of this field relating to mitochondria and cell death is brought out by consideration of studies carried out in mammalian and yeast cells.
Copyright 2002 National Science Council, ROC and S. Karger AG, Basel