Protein ubiquitination requires the concerted action of three enzymes: ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3). These ubiquitination enzymes belong to an abundant protein family that is encoded in all eukaryotic genomes. Describing their biochemical characteristics is an important part of their functional analysis. It has been recognized that there are different E2/E3 specificities and that the detection of E3 ubiquitination activity in vitro may depend on the recruitment of different E2s. Here, we describe the development of an in vitro ubiquitination system based on proteins encoded by genes originally from Arabidopsis. It includes most varieties of Arabidopsis E2 proteins by testing with several RING (Really Interesting New Gene) finger type E3 ligases. This system permits the determination of E3 activity in combination with most of the E2 subgroups that have been identified in the Arabidopsis genome. At the same time, E2/E3 specificities have also been explored. Furthermore, the components employed in this system are all from plants, especially from Arabidopsis, rendering it most suitable for ubiquitination assays of plant proteins. Some E2 proteins which were very difficult to be expressed in E. coli were transiently expressed and purified from plants and then were applied to conquer the problem. This system is also adaptable to proteins of species other than plants. In this system we also provide two different mutated forms of ubiquitin, K48R, and K63R respectively, for detecting different type of ubiquitin conjugation.

Keywords:Ubiquitination;ubiquitin conjugating enzyme;ubiquitin ligase;E2/E3 specificity;in vitro assay;ubiquitin