拟南芥E3连接酶AT2G02960在大肠杆菌中的功能分析

Fuctional Analysis of an Arabidopsis thaliana E3 Ligase At2g02960 in E.coli

摘要:

大肠杆菌处于高温胁迫时，细胞体内激发热激反应，由热休克转录因子σ32 调控多种热激蛋白合成。DnaK/J/E分子伴侣系统在该反应中起着重要作用，包括蛋白质折叠、解聚、水解及跨膜运输等。本研究以一个含有C4HC3锌指的RING结构域蛋白基因AT2G02960（简称ATPRF1）为研究对象，Western Blot检测ATPRF1能够大幅度提高σ32表达水平并稳定σ32，提高大肠杆菌的热耐受性，细菌双杂交及免疫共沉淀实验表明ATPRF1与σ32及DnaK存在相互作用，促进DnaK表达，体外泛素实验验证ATPRF1将σ32泛素化，相互作用位点为RING-finger结构域。我们的研究验证了ATPRF1参与DnaK/J/E分子伴侣体系，提升大肠杆菌的热耐受性并能将σ32多泛素化。

Abstract:

The heat shock response (HSR) in E. coli acts as the cellular response to temperature increase stress. When E. coli is stressed with high temperatures, a series of proteins are synthesized, these proteins are regulated by σ32. The DnaK/J/E chaperone machine of E. coli is essential for the HSR, and involved in protein folding and disaggregation, protelysis and transmembrane transport. In this paper, the AT2G02960 (ATPRF1) protein is a RING-finger E3 ubiquitin ligase in Arabidopsis thaliana. According to the analysis of the growth curve of E. coli, ATPRF1 increased thermotolerance of E. coli. Western Blotting analysis indicated that ATPRF1 could raise the posttranslational levels of heat shock factor σ32 and heat shock protein DnaK, stabilize heat shock factor σ32 simultaneously. Co-immunoprecipitation and bacteria two-hybrid system experiments demonstrated that ATPRF1 interacted with both σ32 and DnaK. σ32 could be ubiquitinated by ATPRF1 in vitro. Hence, our results confirmed that ATPRF1 interacted with DnaK/J/E chaperone and enhanced the thermotolerance of E. coli and ubiquitinated σ32.