BAX-dependent transport of cytochrome c reconstituted in pure liposomes
BAX-dependent transport of cytochrome c reconstituted in pure liposomes
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DOI：
10.1038/35019596
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发表时间：
2000-08-01
影响因子：
21.3
通讯作者：
Schlesinger, PH
中科院分区：
文献类型：
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作者：
Saito, M;Korsmeyer, SJ;Schlesinger, PH
* 华盛顿大学医学院生理学和细胞生物学系，圣路易斯，密苏里州 63110，美国† Dana Farber 癌症研究所，霍华德休斯医学研究所，哈佛医学院，波士顿，马萨诸塞州 02115，美国‡ 电子邮件：paul@cellbio。乌斯特尔。 edu 选出的死亡信号激活促凋亡 BAX，导致易位至线粒体，并作为完整的寡聚膜蛋白插入1-3。随着细胞色素 c 的释放和 caspase4-7 的激活，线粒体功能障碍随之而来。 BAX 在平面脂质双层中形成离子传导通道，并从人工脂质体中释放氯化物或羧基荧光素8、9。BAX 形成的离子传输孔可以发展到约 1.5 nS 的电导，且离子选择性较低。在一些提出的模型中10, 11，BAX与其他成孔分子相互作用，或者通常破坏线粒体外膜10, 11。在这里，我们表明纳摩尔BAX快速形成膜孔12, 13，释放囊泡内羧基荧光素、荧光素-异硫氰酸酯缀合的葡聚糖（FITC-葡聚糖）或FITC-细胞色素c，并且被葡聚糖阻断确定大小的分子。我们还证明了从由两个 BAX 分子组成的 10.7±3.2 Å 孔到需要四个 BAX 分子的 22.1±3.9 Å 之一的浓度依赖性进展，并表明这个较大的 BAX 孔能够运输细胞色素 c。我们使用重组、纯化、功能相同的 BAX（Δ C19）在 pH 7.2 下单分散的分子（参考文献 9、14-17）。先前对蜂毒肽孔的研究表明，指数去猝灭反映了寡聚孔的浓度依赖性激活12、16-19。 BAX 孔激活的指数动力学由方程 (1) 很好地描述，并且拟合度并未通过进一步的指数项得到改善（参见方法）：
* Department of Physiology and Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA† Dana Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA‡ e-mail: paul@ cellbio. wustl. edu elected death signals activate pro-apoptotic BAX, resulting in translocation to mitochondria where it is inserted as an integral, oligomeric membrane protein1–3. Mitochondrial dysfunction follows, with release of cytochrome c and activation of caspases4–7. BAX forms ion-conducting channels in planar lipid bilayers and releases chloride or carboxyfluorescein from artificial liposomes8, 9. The ion-transmitting pore formed by BAX can progress to a conductance of~ 1.5 nS with low ion selectivity. In some proposed models10, 11, BAX interacts with other pore-forming molecules or, alternatively, generally disrupts the outer mitochondrial membrane10, 11. Here we show that nanomolar BAX rapidly forms membrane pores12, 13 that release intravesicular carboxyfluorescein, fluorescein-isothiocyanate-conjugated dextran (FITC–dextran) or FITC–cytochrome c, and that are blocked by dextran molecules of defined size. We also demonstrate concentrationdependent progression from a pore of 10.7±3.2 Å, consisting of two BAX molecules, to one of 22.1±3.9 Å, requiring four BAX molecules, and show that this larger BAX pore is capable of transporting cytochrome c.We analyzed BAX-mediated release of 6-carboxyfluorescein from 200-nm unilamellar vesicles, using recombinant, purified, functionally identical BAX (∆ C19) molecules that were monodispersed at pH 7.2 (refs 9, 14–17). Previous studies of the melittin pore have shown that exponential dequenching reflects concentration-dependent activation of oligomeric pores12, 16–19. The exponential kinetics of BAX-pore activation are well described by equation (1), and the fit was not improved by further exponential terms (see Methods):