A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics
A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics
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DOI：
10.3791/61620
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发表时间：
2020-09-01
影响因子：
1.2
通讯作者：
Chao, Luke H.
中科院分区：
文献类型：
--
作者：
Ge, Yifan;Boopathy, Sivakumar;Chao, Luke H.
线粒体动力学对细胞器的多种功能和细胞反应至关重要。拥挤，空间复杂，线粒体膜是一个具有挑战性的环境，以区分调节因子。蛋白质和脂质成分的实验控制可以帮助回答特定的调节问题。然而，在细胞分析中，这些因素的定量操作是具有挑战性的。为了研究线粒体内膜融合的分子机制，我们引入了一个模拟线粒体内膜脂质环境的体外重构平台。在这里，我们描述了制备脂质双分子层和重建线粒体膜蛋白的详细步骤。该平台允许以定量的方式分析线粒体内膜融合的中间体，以及个体过渡的动力学。本协议描述了不对称脂质组成双分子层的制造，并描述了将跨膜蛋白重构为缓冲双分子层的一般考虑。该方法可应用于其他膜体系的研究。
Mitochondrial dynamics is essential for the organelle's diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.