Yano M, Terada K, Gotoh T, Mori M
Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan.
Exp Cell Res. 2007 Oct 15;313(17):3767-78. doi: 10.1016/j.yexcr.2007.07.031. Epub 2007 Aug 7.
Anti-apoptotic Bcl-2 localizes in the membranes of mitochondria and endoplasmic reticulum (ER) and resists a broad range of apoptotic stimuli. However, the precise function of Bcl-2 in ER is still unclear. We herein examined the anti-apoptotic potencies of Bcl-2 in mitochondria and ER in vitro. The mitochondria isolated from HeLa cells, which have little or practically no Bcl-2, were apoptosis-competent. That is, membrane-bound Bax was activated and cytochrome c was released when the isolated mitochondria were incubated at 35 degrees C. Cytochrome c release from the apoptosis-competent mitochondria was suppressed by co-incubation with the mitochondria with overexpressed Bcl-2 (Bcl-2 mitochondria), suggesting that Bcl-2 anchored in one mitochondrion can suppress cytochrome c release from another mitochondrion. Similar results were obtained when microsomes with overexpressed Bcl-2 (Bcl-2 microsomes) were co-incubated with apoptosis-competent mitochondria. A quantitative titration analysis showed that Bcl-2 in the ER suppresses cytochrome c release as efficiently as that in the mitochondria. An immunoprecipitation assay showed that Bcl-2 in both mitochondria and ER binds to Bax at almost the same degree. However, in the presence of tBid, co-incubation of apoptosis-competent mitochondria with Bcl-2 microsomes, but not with Bcl-2 mitochondria, diminished the Bax-binding to Bcl-2 significantly, suggesting that Bcl-2 in ER is readily inactivated by tBid. Co-incubation assay further confirmed that Bcl-2 in the ER, but not Bcl-2 in the mitochondria, is potentially inactivated by tBid. Our quantitative in vitro studies indicate that Bcl-2 in mitochondria and ER are similarly potent in inhibiting Bax-associated apoptosis of other mitochondria, but are regulated by tBid differently.
抗凋亡蛋白Bcl-2定位于线粒体和内质网（ER）膜上，可抵抗多种凋亡刺激。然而，Bcl-2在内质网中的具体功能仍不清楚。我们在此研究了Bcl-2在线粒体和内质网中的体外抗凋亡能力。从几乎没有或实际上没有Bcl-2的HeLa细胞中分离出的线粒体具有凋亡能力。也就是说，当分离的线粒体在35℃孵育时，膜结合的Bax被激活，细胞色素c被释放。与过表达Bcl-2的线粒体（Bcl-2线粒体）共同孵育可抑制具有凋亡能力的线粒体释放细胞色素c，这表明锚定在一个线粒体中的Bcl-2可以抑制细胞色素c从另一个线粒体中释放。当过表达Bcl-2的微粒体（Bcl-2微粒体）与具有凋亡能力的线粒体共同孵育时，也得到了类似的结果。定量滴定分析表明，内质网中的Bcl-2抑制细胞色素c释放的效率与线粒体中的Bcl-2相同。免疫沉淀试验表明，线粒体和内质网中的Bcl-2与Bax的结合程度几乎相同。然而，在存在tBid的情况下，具有凋亡能力的线粒体与Bcl-2微粒体共同孵育，而不是与Bcl-2线粒体共同孵育，会显著降低Bax与Bcl-2的结合，这表明内质网中的Bcl-2很容易被tBid灭活。共同孵育试验进一步证实，内质网中的Bcl-2而不是线粒体中的Bcl-2可能被tBid灭活。我们的定量体外研究表明，线粒体和内质网中的Bcl-2在抑制其他线粒体的Bax相关凋亡方面同样有效，但受tBid的调节方式不同。
