| GAS2,the beta-subunit of glucosidase II,interacts with the catalytic a-subunit of glucosidase II and removes a-1,3-linked glucose from newly synthesized glycoproteins to generate monoglucosylated core oligosaccharides,which are recognized by chaperone proteins calnexin and calreticulin.As chaperonins in endoplasmic reticulum,calnexin and calreticulin are essential for protein maturation.Our previous study found that OsGAS2 is related to the programmed cell death(PCD).In this study,we explore the,relationship between GAS2 and endoplasmic reticulum stress-mediated PCD pathway in OsGAS2.Transfecting OsGAS2 labeled with GFP into rice protoplasts shows that OsGAS2-GFP co-localized with endoplasmic reticulum,which demonstrates that OsGAS2 localizes on endoplasmic reticulum.The qRT-PCR results suggest that the expression of OsGAS2 in rice suspension cells reduces after treated with an ER Stress inducer DTT.In addition,heat-shock of rice suspension cells,treating rice seedings with low temperature,high salt and drought stress,respectively,reduced the expression of OsGAS2.Taken together,OsGAS2 may play an important role in ER Stress.RNAi-OsGAS2 rice suspension cell lines were established by LH-FAD2-1390 rice interference vector.In RNAi-OsGAS2 rice suspension protoplasts,the cytoplasm Ca2+levels significantly increased after treated with DTT compared with the wild-type(WT)protoplasts.Staining the WT and RNAi-OsGAS2 rice suspension protoplasts with LysoTracker Red DND-99 after treated with DTT showed that the number of autophagy lysosome in cytoplasm of RNAi-OsGAS2 protoplast significantly increased compared WT,which was further confirmed by the TEM analysis.Thus,OsGAS2 is a negative regulation factor of autophagy and ERS down-regulates the OsGAS2 expression levels,which increases Ca2+ concentration and further induces autophagy.OsGAS2 was identified during purification of rice caspase-3 like protease,indicating that the activity of Caspase-3 is closely related to OsGAS2.In order to determine whether OsGAS2 can be cleavaged by caspase-3 protease,we designed the experiments of OsGAS2 cleavaged by caspase-3 protease in vitro.Cleaving OsGAS2 with caspase-3 protease in vitro after prokaryotic expression and purification of OsGAS2 showed that OsGAS2 could be cleaved by caspase-3 protease into four segments with different molecular weight.Western blot analysis of these segments demonstrated that OsGAS2 was a substrate of caspase-3 protease.According to the above results,we analysed the subcellular localization of the five possible fragments and found that P1-P3 segments lacked of a C-terminal ER retention signal located in cytoplasm,P4 and P5 located in ER.To explore the relationship between P1-P5 and PCD,experiments of P1-P5 induced expression in vivo were designed.Binary expression vector pTA7002-P1-P5 induced by dexamethasone(DEX)was constructed and transfected into rice mesophyll protoplasts.P1-P5 would overexpress in cytosol after DEX induction.labeled the protoplasts induced by the above five fragments respectively with LysoTracker Red DND-99,no autophagy lysosomes were found.This indicated the cleavage fragment of OsGAS2 lost its regulation of autophagy;After Heat-shock treatment,the protoplasts induced by the above five fragments were cultured for recovery for 6 h,the caspase-3 like activity analysis showed that DEVDase activity was significantly activated in P5 compared with P1-P4 and WT,suggesting the cleavage fragments of OsGAS2 could magnify the activation of caspase-3 like protease.Taken together,our study illustrated that OsGAS2 played an essential role in autophagy and PCD as a converter:on one hand when moderate ER stress occurred,the expression level of OsGAS2 down-regulated,then induced autophagy by enhancing the Ca2+ concentration in the cytoplasm;on the other hand when severe ER stress occurred,the caspase-3 like protease was activated to cleave OsGAS2,and its cleavage fragment P5 can magnify feedback the activity of caspase-3 and promote PCD occurring. |
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