Faculty of Biotechnology, Department of Cellular Molecular Biology, University of Wroclaw, Wroclaw, Poland.
Methods Mol Biol. 2022;2363:183-197. doi: 10.1007/978-1-0716-1653-6_14.
Mitochondria are subcellular organelles with their own genome and expression system, including translation machinery to make proteins. Several independent studies have shown that translation is an essential regulatory step in expression of the plant mitochondrial genome. Thus, the study of mitochondrial translation seems to be crucial for the comprehension of plant mitochondrial biogenesis and maintenance. In organello protein synthesis in isolated mitochondria is a direct method to visualize the translational products of this organellar genetic system. In this method, highly purified, functional mitochondria synthesize proteins in the presence of radiolabeled amino acids, such as methionine, and an energy regeneration system. The labeled, newly synthesized polypeptides are separated by SDS-polyacrylamide gel electrophoresis and are detected by autoradiography. Here we describe the detailed protocol for in organello labeling of translation products that was optimized for mitochondria isolated from rosette leaves and liquid culture seedlings of Arabidopsis thaliana plants.
线粒体是具有自身基因组和表达系统的亚细胞细胞器，包括用于制造蛋白质的翻译机制。几项独立的研究表明，翻译是植物线粒体基因组表达的一个必要的调控步骤。因此，研究线粒体翻译似乎对于理解植物线粒体生物发生和维持至关重要。在分离的线粒体中进行的体外蛋白质合成是直接观察这个细胞器遗传系统翻译产物的方法。在这种方法中，高度纯化、功能正常的线粒体在放射性标记的氨基酸（如蛋氨酸）和能量再生系统的存在下合成蛋白质。标记的新合成多肽通过 SDS-聚丙烯酰胺凝胶电泳分离，并通过放射自显影检测。在这里，我们描述了优化从拟南芥植物的罗勒叶和液体培养幼苗中分离的线粒体的体外标记翻译产物的详细方案。
