Department of Biochemistry and Molecular Biology, and Genetics, Faculty of Veterinary Sciences, University of Extremadura, Cáceres, Spain.
Methods Enzymol. 2024;707:329-366. doi: 10.1016/bs.mie.2024.07.053. Epub 2024 Aug 17.
Mitochondrial protein import and sorting relies on sophisticated molecular machineries or translocases, of which channels are integral. Channels are built upon membrane proteins whose functions are driven by conformational changes. This implies that structural and functional information need to be integrated to gain a deep understanding of their dynamic behavior. Patch-clamp approaches are well suited for this purpose. This chapter provides a detailed description and practical guidance for applying the patch-clamp methodology to the electrophysiological characterization of mitochondrial protein import. Implementing the technique to intact mitochondria, mitoplasts, and reconstituted proteoliposomes, combined with genetically modified yeast strains, expands the scope of these studies. Focused on the TOM, TIM23, and TIM22 translocases, an analysis of the patch-clamp contribution to the field is outlined.
线粒体蛋白的输入和分拣依赖于复杂的分子机制或转运体，其中通道是组成部分。通道是由膜蛋白构建的，其功能由构象变化驱动。这意味着需要整合结构和功能信息，以深入了解它们的动态行为。膜片钳技术非常适合用于此目的。本章提供了详细的描述和实际指导，用于应用膜片钳方法学来对线粒体蛋白输入进行电生理学特性分析。将该技术应用于完整的线粒体、线粒体膜蛋白小泡和重建的蛋白脂质体，结合经过基因修饰的酵母菌株，扩展了这些研究的范围。本文集中于 TOM、TIM23 和 TIM22 转运体，概述了膜片钳技术对该领域的贡献。
