Department of Protein Biochemistry, Institute of Life Science, Kurume University, Fukuoka, Japan.
Department of Biological Science, Graduate School of Science, Osaka University, Osaka, Japan.
Methods Mol Biol. 2020;2159:115-127. doi: 10.1007/978-1-0716-0676-6_9.
Mitochondria are highly dynamic organelles, which move and fuse to regulate their shape, size, and fundamental function. The dynamin-related GTPases play a critical role in mitochondrial membrane fusion. In vitro reconstitution of membrane fusion using recombinant proteins and model membranes is quite useful in elucidating the molecular mechanisms underlying membrane fusion and to identify the essential elements involved in fusion. However, only a few reconstituting approaches have been reported for mitochondrial fusion machinery due to the difficulty of preparing active recombinant mitochondrial fusion GTPases. Recently, we succeeded in preparing a sufficient amount of recombinant OPA1 involved in mitochondrial inner membrane fusion using a BmNPV bacmid-silkworm expression system. In this chapter, we describe the method for the expression and purification of a membrane-anchored form of OPA1 and liposome-based in vitro reconstitution of membrane fusion.
线粒体是高度动态的细胞器，通过运动和融合来调节其形状、大小和基本功能。与动力蛋白相关的 GTP 酶在线粒体膜融合中起着关键作用。使用重组蛋白和模型膜体外重建膜融合对于阐明膜融合的分子机制以及鉴定融合所涉及的基本要素非常有用。然而，由于难以制备活性的重组线粒体融合 GTP 酶，因此仅报道了几种用于线粒体融合机制的重建方法。最近，我们成功地使用 BmNPV 杆状病毒-家蚕表达系统制备了足够量的参与线粒体内膜融合的重组 OPA1。在本章中，我们描述了膜锚定形式的 OPA1 的表达和纯化以及基于脂质体的体外膜融合重建方法。
