Function Of PRKCSH In The Endoplasmic Reticulum Mediated Programmed Cell Death In Rice

Programmed cell death(PCD)in plants and animals is prevalent in progress of growth and development.Recently,there are three pathways in PCD:death receptor pathway,mitochondrion pathway and endoplasmic reticulum pathway.But the mechanism of the endoplasmic reticulum pathway is still unclear.Our previous study found that PRKCSH is related to PCD.PRKCSH as protein kinase C substrate 80K-H,encodes the beta-subunit of glucosidase ?,which interacts with catalytic ?-subunit of glucosidase ?.Glucosidase ?catalytic removed ?-1,3 N-terminal glucose.We analyse the subcellular localization of PRKCSH and relationship between PRKCSH and endoplasmic reticulum pathway.We analyse cDNA sequences and protein structure of PRKCSH by bioinformatics method:the CDs length of PRKCSH is 1845bp,PRKCSH protien encodes 614 amino acid residues;including the structures of LDLa(Low-density lipoprotein receptor domain class A)?PRKCSH(protein kinase C substrate 80K-H)and N-terminal signal peptide.According to the analysis of subcellular localization prediction and C-terminal HDEL sequence,we speculate that PRKCSH protein is likely to locate in the endoplasmic reticulum.PRKCSH labbed with the GFP is transfected into rice protoplasts.PRKCSH-GFP appeares to co-localize with the blue fluorescences of endoplasmic reticulum dyes.This results suggest that PRKCSH is located at endoplasmic reticulum.With the treatment of DTT,PRKCSH proteins appear spotty phenomena.To further explore the role of PRKCSH in endoplasmic reticulum pathway,we screen proteins interaction with PRKCSH by yeast two-hybrid.First,there are no toxic and autoactivition by identification of pGBKT7-PRKCSH.The recombinant bait vector pGBKT7-PRKCSH fused with rice cDNA library was screened.Primarily,21 positive clones were obtained from SD/-Leu/-Trp/-His/-Ade plates.The 21 positive clones are cultured on SD/-Leu/-Trp/-His/-Ade/X-?-gal/AbA plates,selected 10 positive clones interacting with PRKCSH,obtained 5 unique genes by sequencing analysis.4 positive clones are obtained after verification,4 positive clones:FPP synthase?GFA2?RP L23?R5P3E.According to the relevant researches,GFA2 in the mitochondria is asscociated with apptosis.PRKCSH and GFA2 interact strongest by authentication.To further explore the relationship between PRKCSH and GFA2,PRKCSH is expressed by the prolaryotic expression.PET28a-PRKCSH is transfected into E.coli DE-3,positive clones cultured amplificatively are inducted by IPTG,and two proteins are observed.By sonication on ice,6His-PRKCSH proteins are inclusion bodys.The 6His-PRKCSH proteins are denatured by urea joined with LE Buffer cotainning 0.5mM L-Arg,1mM GSSG and 2mM GSH.Proteins separated and purified by Ni-NTA.Two proteins approximately 80kDa and 110kDa are purified.Two proteins are identified by mass spectrometry,the result reveals that those two proteins are the same PRKCSH protein.Western shows that the 110kDa protein is complete PRKCSH protein.Products of prokaryotic expression play a fundamental role in mechanism between PRKCSH and GFA2.From what has discussed above,we can come to a conclusion that PRKCSH is located in endoplasmic reticulum,PRKCSH and GFA2 interact.As GFA2 in relation to PCD,PRKCSH also is associated with PCD.Products of prokaryotic expression play a fundamental role in the mechanism between PRKCSH and GFA2.In conclusion,PRKCSH proteins maybe involve in ER-mediated PCD.