Screening Of New ERQC Components By Activation Tagging

Whether a protein can function depends on its correct conformation and sub-cellular localization.There exists fine protein quality control machinery in the endoplasmic reticulum(ERQC)to assist newly synthesized peptides folding correctly and then transporting them to secrete.Meanwhile,ERQC machinery can also recognize the misfolded proteins and guide them to degrade through ER-associated degradation(ERAD)pathway.Therefore,ERQC and ERAD play important roles in maintaining the ER homeostasis.Until now,the identified mutants defective in ERQC and ERAD exhibit two extreme phenotypes in Arabidopsis,i.e.,embryonic lethal and wild typelooking under normal growth condition.Both of these phenotypes are not conducive to the cloning and identification of functional genes.The bri1-5(C69Y),the mutated variant of brassinosteroid-sensitive 1(BRI1)has been reported as an ER-residential but functionally competent receptor kinase.It is retained in the ER and degraded by ERAD,resulting in the dwarf phenotype.Therefore,bri1-5 is an ideal substrate for the study of ERQC and ERAD in plant.In current study,the bri1-5 plants were used as experimental material to identify the new components of ERQC and ERAD by activation tagging method.In this project,a total of 726 lines were selected for mutant library.According to phenotype,these lines were classified into three groups,enhanced(more wrinkled leaves),restored(similar to wild type-looking)and non-changed type(similar to bri1-5 mutant).Among these,the T-DNA insertion site of an enhanced line,BE2-53,was identified by adapter-PCR,and the transcript of gene near the insertion site was detected by RT-PCR.The results showed that the expression of a downstream gene was upregulated,named EOB1(enhancer of bri1-5).The overexpression of EOB1 in bri1-5was reminiscent of the dwarf and compact leaves phenotype,implying that the upregulation of the EOB1 is a major factor aggravating dwarf phenotype of bri1-5.The expression pattern and subcellular localization analysis showed that EOB1 was mainly expressed in the vigorous young tissues and located near the ER network.The EOB1 gene encodes a de-ubiquitination protein and the in vitro biochemical assay confirmed its activity.It is possible that the up-regulation of EOB1 may enhance the deubiquitination of key components involved in the ERAD and thus positively regulates the degradation of bri1-5.In addition,the response of eob1 to salt stress was also analyzed and found that eob1 exhibited a similar salt sensitive phenotype as those elements involved in ERQC.In conclusion,a new component of ERAD was screened by activation tagging method and the degradation of bri1-5 protein could be enhanced by ERAD element through de-ubiquitination.