An Quick and Efficient Assay for In Vivo Protein Ubiquitination

doi:10.11983/CBB19153

Abstract

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

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.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB19153

https://www.chinbullbotany.com/EN/Y2019/V54/I6/753

Figures/Tables 7

Figure 1 HY5 and COP1 interact with each other in Nicotiana benthamiana (Liu et al., 2010) (A) Samples were immunoprecipitated with anti-MYC antibody and immunoblotted with anti-GFP antibody; (B) Samples were immunoprecipitated with anti-GFP antibody and immunoblotted with anti-MYC antibody. The numbers on the left show the molecular masses of marker proteins in kiloDaltons (kDa). IgG H: IgG heavy chains

Figure 2 Polyubiquitinated MYC-HY5 and MYC-ELF3 can be detected in Nicotiana benthamiana (Liu et al., 2010) (A) Immunoblots of immunoprecipitated samples with anti- MYC antibody; (B) Immunoblots of immunoprecipitated samples with anti-Ub antibody. The numbers on the left show the molecular masses of marker proteins in kiloDaltons (kDa).

Figure 3 Effects of MG132 on protein stabilities of substrates in Nicotiana benthamiana (Liu et al., 2010) (A) The effect of MG132 on protein stability of HY5-GFP; (B) The effect of MG132 on protein stability of HA-ELF3

Figure 4 COP1-promoted ELF3 degradation can be detected in Nicotiana benthamiana (Liu et al., 2010) * Unspecific bands detected by anti-FLAG antibody.

Figure 5 Detection of COP1-promoted HY5 degradation in a semi-in vivo experiment (Liu et al., 2010) ★ The degradation form of HY5-GFP. The numbers on the left show the molecular masses of marker proteins in kiloDaltons (kDa).

Figure 6 MG132 inhibition on COP1 promote HY5 degradation can be detected in a semi-in vivo experiment (Liu et al., 2010) ★ The degradation form of HY5-GFP. The numbers on the left show the molecular masses of marker proteins in kiloDaltons (kDa).

Figure 7 Proteins expressed in tobacco can be used for in vitro ubiquitination reaction (Liu et al., 2010) (A) Immunoblots of the in vitro ubiquitination assays samples with anti-GFP antibody; (B) Immunoblots of the in vitro ubiquitination assays samples with Nickel-HRP to detect His-ubiquitin. ★ The mixture of poly-ubiquitinated HY5-GFP and Myc-COP1. ▲ Mono-ubiquitinated E2. The numbers on the left show the molecular masses of marker proteins in kiloDaltons (kDa). IgG H: IgG heavy chain

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