The Establishment of a Plant Specific in vitro Ubiquitination Analysis System

Ubiquitination is an important mechanism for regulating most aspects of cell physiology in eukaryote. Adduction of ubiquitin to substrate proteins needs the concerted action of three enzymes: ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3). These enzymes belong to an abundant protein family. It has been recognized that there are large amount of specific combinations of different E2/E3 proteins.

To describe the biochemical characteristics of these ubiquitination enzymes in plants which are an important part of their functional analysis, scientists in Dr. Qi Xie’s group from the Institute of Genetics and Development Biology, the Chinese Academy of Sciences developed a plant specific in vitro ubiquitination system based on proteins mainly expressed from bacteria (Some proteins difficult to be gained from E. coli were transiently expressed and purified from plants).

The components of this system include E1, Ub and the most varieties of Arabidopsis E2 proteins which have been tested with several RING (Really Interesting New Gene) finger type E3 ligases. To detect different types of ubiquitin conjugation, two different mutated forms of ubiquitin, K48R, and K63R are also provided in this system.

Using this system one putative E3’s activity can be detected by combination with most categories of the Arabidopsis E2 members. At the same time, E2/E3 specificities have also been explored.

The components of this system are all from plants, especially from Arabidopsis, rendering it most suitable for ubiquitination assays of plant proteins. This system is also adaptable to proteins of species other than plants.

This work with the graduate student Qingzhen Zhao as the first author has been online published on The Plant Journal (doi: 10.1111/tpj.12127). This research was supported by grants from the Ministry of Science and Technology, National Natural Science Foundation of China and the Chinese Academy of Sciences.