Department of Plant Physiology and Protein Metabolism Laboratory, University of Osnabruck, Osnabruck, Germany; CellNanOs-Center of Cellular Nanoanalytics, University of Osnabruck, Osnabruck, Germany; ScienceCampus Halle-Plant-Based Bioeconomy, Halle (Saale), Germany.
Department of Plant Physiology and Protein Metabolism Laboratory, University of Osnabruck, Osnabruck, Germany; CellNanOs-Center of Cellular Nanoanalytics, University of Osnabruck, Osnabruck, Germany; ScienceCampus Halle-Plant-Based Bioeconomy, Halle (Saale), Germany.
Methods Enzymol. 2023;686:205-220. doi: 10.1016/bs.mie.2023.02.014. Epub 2023 Mar 24.
As a part of the ubiquitin-proteasome system, E3 ubiquitin ligases play an important role in the regulation of the proteome in eukaryotic cells. These enzymes are extensively studied because of their crucial function, however it can be challenging to observe E3 ubiquitin ligases in action. Here, we outline a method for determining whether a known or potential E3 ubiquitin ligase exhibits autoubiquitination activity in vitro using PROTEOLYSIS1 (PRT1, AT3G24800), the first identified N-degron pathway E3 ubiquitin ligase from plants as an example. The approach provided here makes it possible to analyze mutations that could reduce or eliminate activity, to test for interaction with E2 ubiquitin conjugating enzymes, as well as to check for in vitro substrate ubiquitination.
作为泛素-蛋白酶体系统的一部分，E3 泛素连接酶在真核细胞的蛋白质组调控中发挥着重要作用。这些酶因其关键功能而被广泛研究，但观察 E3 泛素连接酶的作用具有一定挑战性。在这里，我们以植物中首个被鉴定的 N 端降解途径 E3 泛素连接酶 PROTEOLYSIS1（PRT1，AT3G24800）为例，概述了一种使用体外实验来确定已知或潜在 E3 泛素连接酶是否具有自身泛素化活性的方法。本研究提供的方法可用于分析可能降低或消除活性的突变，测试与 E2 泛素连接酶的相互作用，以及检查体外底物的泛素化。
