Sato Takeo, Sako Kaori, Yamaguchi Junji
Faculty of Science and Graduate School of Life Science, Hokkaido University, Kita-ku, Sapporo, Japan.
Methods Mol Biol. 2014;1072:655-63. doi: 10.1007/978-1-62703-631-3_45.
The ubiquitin-26S proteasome system (UPS) plays a crucial role in selective removal of short-lived target proteins, archiving fine-tuning of post-translation levels of the target proteins. Recently a number of ubiquitin ligases (E3) have been reported as essential regulators of various plant developmental cues and stress responses. To clarify the detailed biochemical and physiological function of the E3 proteins, identification of their target proteins is of great importance. A transient expression system with tobacco leaves is a powerful method to evaluate E3 function and target degradation via UPS. Here simple methods to assay proteasome-dependent protein degradation combined with a tobacco transient expression system and detection of accumulation of ubiquitinated proteins are presented.
泛素-26S蛋白酶体系统（UPS）在选择性清除短命靶蛋白、对靶蛋白翻译后水平进行精细调控方面发挥着关键作用。最近，许多泛素连接酶（E3）已被报道为各种植物发育线索和应激反应的重要调节因子。为了阐明E3蛋白详细的生化和生理功能，鉴定其靶蛋白非常重要。烟草叶片瞬时表达系统是评估E3功能以及通过UPS进行靶蛋白降解的有力方法。本文介绍了结合烟草瞬时表达系统来检测蛋白酶体依赖性蛋白降解以及检测泛素化蛋白积累的简单方法。
