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

作　　者：Huihui Liu Simiao Liu Hong Yu Xiahe Huang Yingchun Wang Liang Jiang Xiangbing Meng Guifu Liu Mingjiang Chen Yanhui Jing Feifei Yui Bing Wang Jiayang Li 

机构地区：[1]State 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 [2]University of Chinese Academy of Sciences,Beijing 100049,China

出　　处：《Molecular Plant》2022年第8期1285-1299,共15页分子植物（英文版）

基　　金：The research was supported by grants from the National Natural Science Foundation of China(32133012,31788103,32100268,and 31900244);the Youth Innovation Promotion Association of the Chinese Academy of Sciences(2019099).

摘　　要：Multisubunit SKP1/Cullin1/F-box(SCF)E3 ligases play essential roles in regulating the stability of crucial regulatory factors and controlling growth and development in eukaryotes.Detecting E3 ligase activity in vitro is important forexploring the molecular mechanism of protein ubiquitination.However,in vitro ubiquitination assay systems for multisubunit E3 ligases remain difficult to achieve,especially in plants,mainly owing to difficulties in achieving active components of multisubunit E3 ligases with high purity and characterizing specific E2 and E3 pairs.In this study,we characterized components of the rice ScFDiwARF3(SCFDs)E3 ligase,screened the coordinated E2,and reconstituted active ScFD3 E3 ligase in vitro.We further engineered SCFD3 E3 ligase using a fused SKP1-Cullin1-RBX1(eSCR)protein and found that both the wild-type SCFD3 E3 ligase and the engineered SCFD3 E3 ligase catalyzed ubiquitination of the substrate D53,which is the key transcriptional repressor in strigolactone signaling.Finally,we replaced D3 with other F-box proteins from rice and humans and reconstituted active escF E3 ligases,including escFaID2,escFBxL1s,and escFcDC4 E3 ligases.Our work reconstitutes functional SCF E3 ligases in vitro and generates an engineered system with interchangeable F-box proteins,providing a powerful platform for studying the mechanisms of multisubunit SCF E3 ligases in eukaryotes.

关 键 词：RICE SCF E3 ligase UBIQUITINATION STRIGOLACTONES DWARF53 Sic1 

分 类 号：Q94[生物学—植物学]