Lim Maria L R, Lum May-Ghee, Hansen Tania M, Roucou Xavier, Nagley Phillip
Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Vic., Australia.
J Biomed Sci. 2002;9(6 Pt 1):488-506. doi: 10.1159/000064722.
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.
线粒体在细胞凋亡中发挥关键作用，其中一个核心步骤是细胞色素c（cyt c）跨越线粒体外膜释放到细胞质中。我们从诱导线粒体释放cyt c的影响因素、这种释放的可能机制以及半胱天冬酶激活的下游后果等方面对这一过程进行综述。我们还会考虑Bcl-2家族成员在调节与凋亡信号相关的线粒体活动中的作用。抗凋亡成员，如Bcl-2本身，与其他家族成员相互拮抗，在众多与Bcl-2同源的促凋亡蛋白中，Bax尤为突出。重点介绍了测定cyt c释放的技术方法，包括细胞分级分离、生物化学、免疫化学和共聚焦显微镜检查[包括使用cyt c-绿色荧光蛋白（GFP）融合蛋白实时观察释放情况]。讨论了各种方法的优点和潜在缺陷，特别强调了cyt c-GFP融合蛋白的使用以及对cyt c和其他具有促凋亡功能的线粒体蛋白可能通过的假定外膜孔特征的测定。通过对哺乳动物细胞和酵母细胞的研究可以看出，这个与线粒体和细胞死亡相关的领域内容丰富。
