The Molecular Mechanism Of Ubiquitin Ligase Mf630 Involved In Endoplasmic Reticulum Stress

RING-finger E3 ubiquitin ligase is located in endoplasmic reticulum membrane and involved in endoplasmic reticulum-associated degradation(ERAD).ERAD is the process in which misfolded proteins of endoplasmic reticulum lumen are transferred to the cytoplasm and degraded by ubiqutination.ERAD is a key process for endoplasmic reticulum-mediated protein quality control(ERQC).ERAD is activated by ER stress,which maintances the function of ER and responses to stress.Previous studies regarding ERAD-related E3 ligase were mainly focused on yeast and animal,while little is known in plants.Besides,the limited number of ERAD-related E3 in plants are homology of yeast and animal,plant-specific ERAD-related E3 ligase haven't been reported.Based on transcriptome analysis of Medicgao falcata,A RING-finger E3 ubiquitin ligase gene,Mf630,which upregulated by salt and drought was identified.qRT-PCR and Promoter-GUS staining assay showed that Mf630 expressed in the root and leaves and is induced by drought,salt and tunicamycin.Cell fractionation assays result indicated Mf630 is a membrane protein.Transient expression of Mf630-GFP in Arabidopsis protoplast revealed that Mf630 located in endoplasmic reticulum membrane.In addition,in vitro ubiquitination assay demonstrated that Mf630 displayed ubiquitin ligase activity.The roots of overexpressing Mf630 trangenic Arabidopsis plants were longer than WT under salt treatment.Besides,compare to WT,more cotyledon of Mf630 trangenic Arabidopsis plants exhibited green under tunicamycin treatment and longer.These results suggested that Mf630 was involved in endoplasmic reticulum stress,and enhance Tm and salt tolerance of transgenic Arabidopsis.We then studied the function of Mf630 in Medicgao truncatula,the etiolation rate of Mf630 overexpression lines was lower than WT under salt stress,RNAi line of Mt630 and mt630 exhibited in the opposite way,which indicating Mf630 is necessary for salt tolerance of Medicgao truncatula.In order to study the mechanism of Mf630 involving salt tolerance of Medicago truncatula,a split-ubiquitin system method was used to screen interacting protein.We identified three candidate proteins,MtSec6ly,MtUBC32 and MtUBC34,which located in ER membrane.MtSec6ly is a subunit of the Sec61 protein translocation channel at the endoplasmic reticulum and Sec61 complex is shown to participate in the transport of the ERAD substrate.MtUBC32 and MtUBC34 were predicted to be ubiquitin-conjugating enzyme of endoplasmic reticulum,and their homologous gene in yeast was also involved in ERAD.Bimolecular fluorescence complementation assays and Firefly luciferase complementation assays confirmed the interaction between Mf630 and MtSec6l?,MtUBC32 and MtUBC34.Further studies showed that MtSec61? was not the substrate of Mf630.So we hypothesized Mf630 interacts with Sec61 complex through MtSec61? and ubiquitinates ERAD-related substrates which transported by Sec61,MtUBC32 or MtUBC34 may involve in this degradation process as an ubiquitin-conjugating enzyme.Semi-in vivo degradation experiment of Mf630-HA and AtCPY*-GFP showed that Mf630 promoted the degradation of AtCPY*-GFP,the known ERAD substrate.These data indicated that Mf630 was involved in Sec61-related ERAD process.Taken together,we found a novel RING-finger ubiquitin E3 ligase,Mf630,which enhanced salt tolerance of Medicago.Mf630 located in the endoplasmic reticulum and might participate in ERAD through the interactions with MtSec61,MtUBC32 and MtUBC34.We hypothesize that under salt stress,misfolded proteins transported by the Sec61 complex are recognized by Mf630 through the interaction between Mf630 and the Sec61y subunit,and then ubiquinated via UBC32 or UBC34 for degradation.This study is the first report of ubiquitin ligase related to the endoplasmic reticulum degradation in plants and could enrich the understanding of the ERAD progress.