Landeta Olatz, Garcia Valero Juan, Flores-Romero Hector, Bustillo-Zabalbeitia Itsasne, Landajuela Ane, Garcia-Porras Miguel, Terrones Oihana, Basañez Gorka
Unidad de Biofisica, Centro Mixto Consejo Superior de Investigaciones Científicas (CSIC)-Euskal Herriko Unibertsitatea/Universidad del Pais Vasco (EHU/UPV) , Barrio Sarriena s/n, Leioa 48940, Spain.
ACS Chem Biol. 2014 Dec 19;9(12):2852-63. doi: 10.1021/cb500592e. Epub 2014 Oct 22.
Increasing evidence indicates that the mitochondrial lipid membrane environment directly modulates the BCL2 family protein function, but the underlying mechanisms are still poorly understood. Here, we used minimalistic reconstituted systems to examine the influence of mitochondrial lipids on MCL1 activity and conformation. Site-directed mutagenesis and fluorescence spectroscopic analyses revealed that the BCL2 homology region of MCL1 (MCL1ΔNΔC) inhibits permeabilization of MOM-like membranes exclusively via canonical BH3-into-groove interactions with both cBID-like activators and BAX-like effectors. Contrary to currently popular models, MCL1ΔNΔC did not require becoming embedded into the membrane to inhibit membrane permeabilization, and interaction with cBID was more productive for MCL1ΔNΔC inhibitory activity than interaction with BAX. We also report that membranes rich in cardiolipin (CL), but not phosphatidylinositol (PI), trigger a profound conformational change in MCL1ΔNΔC leading to membrane integration and unleashment of an intrinsic lipidic pore-forming activity of the molecule. Cholesterol (CHOL) reduces both the conformational change and the lipidic pore-forming activity of MCL1ΔNΔC in CL-rich membranes, but it does not affect the interaction of MCL1ΔNΔC with proapoptotic partners in MOM-like liposomes. In addition, we identified MCL1α5 as the minimal domain of the protein responsible for its membrane-permeabilizing function both in model membranes and at the mitochondrial level. Our results provide novel mechanistic insight into MCL1 function in the context of a membrane milieu and add significantly to a growing body of evidence supporting an active role of mitochondrial membrane lipids in BCL2 protein function.
越来越多的证据表明，线粒体脂质膜环境直接调节BCL2家族蛋白的功能，但其潜在机制仍知之甚少。在这里，我们使用简约的重构系统来研究线粒体脂质对MCL1活性和构象的影响。定点诱变和荧光光谱分析表明，MCL1的BCL2同源区域（MCL1ΔNΔC）仅通过与cBID样激活剂和BAX样效应器的经典BH3-凹槽相互作用来抑制类线粒体外膜（MOM）样膜的通透性。与当前流行的模型相反，MCL1ΔNΔC不需要嵌入膜中就能抑制膜通透性，并且与cBID的相互作用对MCL1ΔNΔC的抑制活性比与BAX的相互作用更有效。我们还报告说，富含心磷脂（CL）而非磷脂酰肌醇（PI）的膜会引发MCL1ΔNΔC的深刻构象变化，导致膜整合并释放该分子固有的脂质成孔活性。胆固醇（CHOL）可降低富含CL的膜中MCL1ΔNΔC的构象变化和脂质成孔活性，但不影响MCL1ΔNΔC与类MOM脂质体中促凋亡伙伴的相互作用。此外，我们确定MCL1α5是该蛋白在模型膜和线粒体水平上负责其膜通透功能的最小结构域。我们的结果为膜环境背景下MCL1的功能提供了新的机制见解，并显著增加了越来越多的证据，支持线粒体膜脂质在BCL2蛋白功能中发挥积极作用。
