Effects Of Methylated Modification And Site Mutation On The Function Of Arabidopsis Protein SEC24 Respectively

Endomembrane system is a typical characteristics of eukaryotic cells,which differs eukaryotic cells from prokaryotic cells. Membrane system is composed of many organelles, each organelle has its own unique components and functions. In order to maintain the dynamic balance of the organelles in morphology and function, exchanges of a large number of membrane fractions and organelle lumen substances between organelles, which are accomplished by protein transport, continuously take place, Among the protein transport processes, transport of proteins from the ER to the Golgi are carried out by COPII vesicles, which consists of five cytoplasmic proteins: Sar1, inner shell component Sec23 and Sec24 and outer cage component Sec13 and Sec31. SEC24 is highly conserved in eukaryotes, it has multiple binding sites for protein cargos distributed in its surface, which interaction with the signal peptide of proteins and recruit them into COPII vesicles.Earlier studies have shown that Arabidopsis contains four SEC24 isoforms(SEC24A-D), among which,point mutation of SEC24 on the 693 th amino acid can cause abnormal morphologies of ER and Golgi.This 693 th amino acid is highly conserved in eukaryotes, and plays an important role in sorting cargo proteins. But what impacts this site mutation has on to protein sorting and transport is unclear. Secondly,Xiaofeng Cao's research team found that SEC24 can be specifically methylated by arginine methyltransferase 4(At PRMT4) on its N-terminal domain,but the impact of methylation modification of SEC24 on its function is not yet known,In order to find answers to these questions, we analysed the total proteins by ITRAQ technique and vacuole proteins by SDS-PAGE follow LC-MS/MS from Atprmt4 mutants and sec24 a mutants respectively, and compared the protein profiles of these mutants with those of wild-type Arabidopsis.By comparing the profile of total proteins in Atprmt4 mutants with that of wild-type arabidopsis, it was found that the differentially expressed proteins in Atprmt4 mutants are mainly located in chloroplasts and plastids, many of these proteins function in photosynthetic electron transport and down-regulated in different degrees. These results suggested that methylation modification of SEC24 may affect the sorting and transport of proteins which are destined to for the chloroplast and plastid membrane. In Atprmt4 mutants, these proteins can not be transported to their destination and are thus degraded. Proteomic results also showed that the differentially expressed proteins in sec24 a mutant, are also mainly located in the chloroplast and the plastid, but many of them function as oxidoreductase and glucosidase in carbon metabolism and amino acid biosynthesis pathways. These pathways takes place in the stroma part of chloroplast, which suggested that mutations in sec24 a gene may affect the sorting and transport of soluble proteins that are destined for the stroma parts of the chloroplast.Vacuoles protein from wild type arabidopsis and mutants were successfully isolated and enriched using ultracentrifugation technique. The vacuole protein analysis results showed that the missing proteins are mostly membrane-bound proteins which from At PRMT4 mutants. but, The missing vacuole protein from sec24 a single base mutant are mostly soluble protein,further confirmed that methylation modification of SEC24 may affect the sorting and transport of membrane-bound proteins and the sec24 a gene may affect the sorting and transport of soluble proteins from the subcellular level.but,lots of further studies are needed to confirm these hypotheses.