Association of Bax and Bak homo-oligomers in mitochondria -: Bax requirement for Bak reorganization and cytochrome c release
Association of Bax and Bak homo-oligomers in mitochondria -: Bax requirement for Bak reorganization and cytochrome c release
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DOI：
10.1074/jbc.m203392200
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发表时间：
2003-02-14
影响因子：
4.8
通讯作者：
Saikumar, P
中科院分区：
文献类型：
--
作者：
Mikhailov, V;Mikhailova, M;Saikumar, P
缺氧或线粒体抑制剂诱导的ATP耗竭导致Bax从细胞质移位到线粒体，并使细胞色素c从线粒体释放到培养的大鼠近曲小管细胞的细胞质中。易位的Bax经历进一步的构象变化以寡聚成高分子量复合物（Mikhailov，V.，Mikhailova，M.，Pulkrabek，D. J.，董，Z. Venkatachalam，M.一、和Saikumar，P.（2001）J.Biol.Chem.276，18361-18374）。在这里，我们报告，Bax易位在ATP耗竭大鼠近端小管细胞，巴克，一个促凋亡分子，通常居住在线粒体，也重组，形成同源寡聚体。Bax和巴克的寡聚化独立于Bid裂解和/或易位发生。化学交联膜提取物的Western印迹显示Bax和巴克复合物的非重叠“阶梯”，分别为21和23 kDa的倍数，与每个阶梯内的分子同质性一致。这表明Bax和巴克复合物是同源寡聚体。尽管如此，每种低聚物都可以与另一种低聚物发生免疫共沉淀，这表明Bax和巴克之间存在一定程度的亲和力，允许共沉淀但不允许交联。此外，通过SDS和免疫沉淀之前的复性交联复合物的解离并不能阻止两种寡聚物的重新结合。值得注意的是，Bcl-2的表达不仅阻止了Bax和巴克的寡聚化，而且阻止了这两种蛋白质在能量剥夺细胞中的结合。使用Bax缺陷的HCT 116和BMK细胞，我们发现，有严格的Bax的要求巴克同源寡聚化和细胞色素c释放过程中的能量剥夺。使用Bak缺陷的BMK细胞，我们进一步表明，巴克缺陷与Bax易位的延迟动力学相关，但不影响易位的Bax的寡聚化或细胞色素c的泄漏。这些结果表明Bax和巴克在这种形式的细胞损伤的功能合作的程度，但也证明了线粒体透化的Bax的绝对要求。
ATP depletion induced by hypoxia or mitochondrial inhibitors results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured rat proximal tubule cells. Translocated Bax undergoes further conformational changes to oligomerize into high molecular weight complexes (Mikhailov, V., Mikhailova, M., Pulkrabek, D. J., Dong, Z., Venkatachalam, M. A., and Saikumar, P. (2001) J. Biol. Chem. 276, 18361-18374). Here we report that following Bax translocation in ATP-depleted rat proximal tubule cells, Bak, a proapoptotic molecule that normally resides in mitochondria, also reorganizes to form homo-oligomers. Oligomerization of both Bax and Bak occurred independently of Bid cleavage and/or translocation. Western blots of chemically cross-linked membrane extracts showed nonoverlapping "ladders" of Bax and Bak complexes in multiples of similar to21 and similar to23 kDa, respectively, consistent with molecular homogeneity within each ladder. This indicated that Bax and Bak complexes were homo-oligomeric. Nevertheless, each oligomer could be co-immunoprecipitated with the other, suggesting a degree of affinity between Bax and Bak that permitted co-precipitation but not cross-linking. Furthermore, dissociation of cross-linked complexes by SDS and renaturation prior to immunoprecipitation did not prevent reassociation of the two oligomeric species. Notably, expression of Bcl-2 prevented not only the oligomerization of Bax and Bak, but also the association between these two proteins in energy-deprived cells. Using Bax-deficient HCT116 and BMK cells, we show that there is stringent Bax requirement for Bak homo-oligomerization and for cytochrome c release during energy deprivation. Using Bak-deficient BMK cells we further show that Bak deficiency is associated with delayed kinetics of Bax translocation but does not affect either the oligomerization of translocated Bax or the leakage of cytochrome c. These results suggest a degree of functional cooperation between Bax and Bak in this form of cell injury, but also demonstrate an absolute requirement of Bax for mitochondrial permeabilization.