Le Boulch Marie, Brossard Audrey, Le Dez Gaëlle, Léon Sébastien, Rabut Gwenaël
Univ Rennes, CNRS, IGDR (Institute of Genetics and Development of Rennes) - UMR 6290, F-35000 Rennes, France.
Institut Jacques Monod - UMR 7592, CNRS, Université de Paris-Diderot, F-75205 Paris Cedex 13, France.
J Cell Sci. 2020 Jun 24;133(12):jcs240093. doi: 10.1242/jcs.240093.
Ubiquitylation is a reversible post-translational protein modification that regulates a multitude of cellular processes. Detection of ubiquitylated proteins is often challenging because of their low abundance. Here, we present NUbiCA, a sensitive protein-fragment complementation assay to facilitate the monitoring of ubiquitylation events in cultured cells and model organisms. Using yeast as a model system, we demonstrate that NUbiCA enables accurate monitoring of mono- and polyubiquitylation of proteins expressed at endogenous levels. We also show that it can be applied to decipher the topology of ubiquitin conjugates. Moreover, we assembled a genome-wide collection of yeast strains ready to investigate the ubiquitylation of proteins with this new assay. This resource will facilitate the analysis of local or transient ubiquitylation events that are difficult to detect with current methods.
泛素化是一种可逆的蛋白质翻译后修饰，可调节多种细胞过程。由于泛素化蛋白丰度低，检测它们往往具有挑战性。在这里，我们介绍了NUbiCA，这是一种灵敏的蛋白质片段互补分析方法，有助于监测培养细胞和模式生物中的泛素化事件。以酵母作为模型系统，我们证明NUbiCA能够准确监测内源性表达蛋白的单泛素化和多泛素化。我们还表明，它可用于解析泛素缀合物的拓扑结构。此外，我们构建了一个全基因组酵母菌株库，准备用这种新方法研究蛋白质的泛素化。该资源将有助于分析目前方法难以检测的局部或瞬时泛素化事件。
