Chinese Bulletin of Botany ›› 2019, Vol. 54 ›› Issue (6): 753-763.DOI: 10.11983/CBB19153

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

An Quick and Efficient Assay for In Vivo Protein Ubiquitination

Lijing Liu1,Qingzhen Zhao2,Qi Xie3,4,Feifei Yu3,*()   

  1. 1 College of Life Sciences, Shandong University, Qingdao 266237, China
    2 College of Life Sciences, Liaocheng University, Liaocheng 252000, 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:2019-09-24 Online:2019-11-01 Published:2020-07-09
  • Contact: Feifei Yu

Abstract:

The UPS (ubiquitination/proteasome system) plays a vital role in nearly all plant signaling processes, for example, jasmonic acid receptor COI1 (coronotine insensitive protein 1) and auxin receptor TIR1 (transport inhibitor response 1) are F-box type E3s ligases, and they promote the ubiquitination then degradation of specific transcriptional repressors through 26S proteasome to activate hormone signaling. However, for the whole UPS system, only a few E3 ligase/substrate pairs’ interactions have been demonstrated due to technical limitations in plants. Generally, E3 ligase and substrate are expressed in Escherichia coli for ubiquitination assay, which may lack post-translational modifications that are important for protein function, then gives false negative result. We describe a quick and efficient assay for detecting E3-mediated protein ubiquitination in vivo by means of agroinfiltration for transient expression of relevant genes in tobacco (Nicotiana benthamiana) leaves. In detail, this method can detect the specific interaction between E3 ligase and substrate, substrate ubiquitination, the effect of E3 ligase on degradation of its substrate, inhibition of substrate degradation by 26S proteasome inhibitor MG132, and in vitro ubiquitination assay with endogenous plant proteins.

Key words: in vivo ubiquitination assay, E3 ubiquitin ligases, transient expression system in tobacco