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

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.