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Related Experiment Video

Updated: Sep 6, 2025

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Published on: December 5, 2019

An engineered platform for reconstituting functional multisubunit SCF E3 ligase in vitro.

Huihui Liu¹, Simiao Liu¹, Hong Yu²

¹State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, The Innovative Academy for Seed Design, Chinese Academy of Sciences, Beijing 100101, China.

Molecular Plant

|June 25, 2022

Summary

Researchers reconstituted active SKP1/Cullin1/F-box (SCF) E3 ligases in vitro, creating an engineered system with interchangeable F-box proteins for studying ubiquitination mechanisms.

Keywords:

DWARF53 SCF E3 ligase Sic1 rice strigolactones ubiquitination

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Last Updated: Sep 6, 2025

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Published on: December 5, 2019

In Vitro Analysis of E3 Ubiquitin Ligase Function

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Published on: May 10, 2022

Area of Science:

Biochemistry
Molecular Biology
Plant Science

Background:

Multisubunit SKP1/Cullin1/F-box (SCF) E3 ligases regulate eukaryotic growth and development by controlling protein stability.
In vitro ubiquitination assays are crucial for understanding protein ubiquitination mechanisms but are challenging for plant SCF E3 ligases due to component purity and E2/E3 pair characterization difficulties.

Purpose of the Study:

To characterize rice SCF^DWARF3 (SCF^D3) E3 ligase components and reconstitute its activity in vitro.
To engineer an SCF^D3 E3 ligase system using a fused SKP1-Cullin1-RBX1 (eSCR) protein.
To develop a versatile platform for studying SCF E3 ligases by enabling interchangeable F-box proteins.

Main Methods:

Characterization of rice SCF^D3 E3 ligase components.
Screening for cognate E2 enzymes.
In vitro reconstitution of active SCF^D3 E3 ligase.
Engineering of SCF^D3 E3 ligase using eSCR protein.
Ubiquitination assays using D53 substrate.
Reconstitution of various eSCF E3 ligases with different F-box proteins.

Main Results:

Active SCF^D3 E3 ligase was successfully reconstituted in vitro.
Both wild-type and engineered SCF^D3 E3 ligases catalyzed the ubiquitination of D53, a key repressor in strigolactone signaling.
Functional eSCF E3 ligases (eSCF^GID2, eSCF^FBXL18, eSCF^CDC4) were reconstituted using interchangeable F-box proteins from rice and humans.

Conclusions:

This study successfully reconstitutes functional SCF E3 ligases in vitro.
An engineered system with interchangeable F-box proteins was developed, offering a powerful platform for studying multisubunit SCF E3 ligase mechanisms in eukaryotes.