Chinese Bulletin of Botany ›› 2019, Vol. 54 ›› Issue (6): 764-772.DOI: 10.11983/CBB19152

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

In Vitro Ubiquitination Assay for Plant Proteins

Qingzhen Zhao1,Lijing Liu2,Qi Xie3,4,Feifei Yu3,*()   

  1. 1 College of Life Sciences, Liaocheng University, Liaocheng 252000, China
    2 College of Life Sciences, Shandong University, Qingdao 266237, China
    3 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
    4 University of Chinese Academy of Sciences, Beijing 100049, China
  • Received:2019-08-12 Accepted:2019-10-31 Online:2019-11-01 Published:2020-07-09
  • Contact: Feifei Yu

Abstract:

Ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3) are the key enzymes of ubiquitin modification of substrate proteins. There are large amounts of genes encoding these ubiquitination enzymes in all eukaryotic genomes. Analyzing the biochemical characteristics and specificity of these enzymes and their substrate proteins is important for their functional study. Here we describe a simple and fast method for in vitro ubiquitination assay. In the presence of E1 and ubiquitin, E2 activity can be determined by detecting the DTT-sensitive thio-ester formation. The E3 activity of a putative protein as well as the E2-E3 or E3-substrate specificities can also be explored by in vitro ubiquitination assay. This system is mainiy based on proteins from Arabidopsis, which includes most varieties of Arabidopsis E2 proteins that are tested with several RING-finger type E3 ligases. This system facilitate not only the exploration of E3 activity in combination with various Arabidopsis E2 members but also the study of E2-RING E3 and RING E3-substrate specificities. This system is suitable for the ubiquitination assays of eukaryotic proteins, especially for plant proteins.

Key words: in vitro ubiquitination assay, ubiquitin conjugating enzyme (E2), ubiquitin protein ligase (E3)