An Quick and Efficient Assay for In Vivo Protein Ubiquitination
Received date: 2019-08-12
Accepted date: 2019-09-24
Online published: 2019-10-09
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.
Lijing Liu,Qingzhen Zhao,Qi Xie,Feifei Yu . An Quick and Efficient Assay for In Vivo Protein Ubiquitination[J]. Chinese Bulletin of Botany, 2019 , 54(6) : 753 -763 . DOI: 10.11983/CBB19153
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