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A plant‐specific in vitro ubiquitination analysis system
Author(s) -
Zhao Qingzhen,
Tian Miaomiao,
Li Qingliang,
Cui Feng,
Liu Lijing,
Yin Bojiao,
Xie Qi
Publication year - 2013
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.12127
Subject(s) - ubiquitin , arabidopsis , ubiquitin ligase , ubiquitin conjugating enzyme , biology , ubiquitin protein ligases , biochemistry , microbiology and biotechnology , enzyme , in vitro , genetics , gene , mutant
Summary 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 various E 2/ E 3 specificities exist, and that detection of E 3 ubiquitination activity in vitro may depend on the recruitment of E 2s. Here, we describe the development of an in vitro ubiquitination system based on proteins encoded by genes from Arabidopsis. It includes most varieties of Arabidopsis E 2 proteins, which are tested with several RING ‐finger type E3 ligases. This system permits determination of E3 activity in combination with most of the E 2 sub‐groups that have been identified in the Arabidopsis genome. At the same time, E 2/ E 3 specificities have also been explored. The components used in this system are all from plants, particularly Arabidopsis, making it very suitable for ubiquitination assays of plant proteins. Some E 2 proteins that are not easily expressed in E scherichia coli were transiently expressed and purified from plants before use in ubiquitination assays. This system is also adaptable to proteins of species other than plants. In this system, we also analyzed two mutated forms of ubiquitin, K 48R and K 63 R , to detect various types of ubiquitin conjugation.