Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo;
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo.
J Vis Exp. 2021 Jul 19(173). doi: 10.3791/62853.
Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.
尽管最近在酵母线粒体蛋白质组的特征描述方面取得了进展，但仍有大量蛋白质的亚线粒体定位难以捉摸。在这里，我们描述了一种用于确定酵母线粒体蛋白质亚细胞器定位的稳健而有效的方法，这被认为是阐明线粒体蛋白质功能的基本步骤。该方法包括一个初始步骤，包括获得高度纯净的完整线粒体。然后将这些线粒体制剂进行亚分级分离方案，包括低渗冲击（肿胀）和用蛋白酶 K（蛋白酶）孵育。在肿胀过程中，选择性地破坏外线粒体膜，允许蛋白酶 K 消化膜间空间隔室的蛋白质。同时，为了获得关于膜蛋白拓扑结构的信息，线粒体制剂最初进行超声处理，然后用碳酸钠进行碱性提取。最后，在离心后，通过 SDS-PAGE 和 Western blot 分析来自这些不同处理的沉淀和上清液部分。通过将目标蛋白质的 Western blot 图谱与已知标准进行比较，获得其亚线粒体定位和膜拓扑结构。
