Functional Research Of Exocyst Complex In The Localization Of Pectin Methylesterase 17 In Arabidopsis

Protein transport between plant cells is accomplished by vesicle trafficking.Tethering is really important for vesicle targeting to the acceptor compartment during the process of vesicle trafficking.Exocyst is an octameric tethering complex,which mainly mediates the tethering process between vesicles and plasma membrane.Studies have shown that exocyst complex participates in the process of plant development and cell morphogenesis,including cytokinesis.Exocyst subunits SEC6,SEC8,EXO70A1,and EX084B located at the cell plate,and are involved in the process of cytokinesis.However,it is unclear how do they actually work.Pectin methylesterase(PME)is ubiquitous enzyme in plant,which can modify the degree of methylesterification of pectin in plant cell wall,then influence plant development.Our primary result of yeast two hybrid assay indicates that PME17 interact with SEC6.Besides,the localization of PME is at the cell plate.In this study,we made use of exocyst mutants(PRsec6,exo70a1,exo70e2 and exo84b)and pro35S:PME17-GFP transgenic plant in Arabidopsis to investigate the function of exocyst complex in the localization of PME 17.We find that PME 17 transport to cell wall and cell plate through secretion,and SEC6 is required during this process.This research suggests that exocyst complex might influence cell plate development by regulating secretion of cell wall structure enzymes,like PME.1.PME 17 is mainly located at cell wall and cell plate,while it participates the whole process of cell plate formationWe found PME 17 is located at cell wall through the observation of 35S:PME17-GFP transgenic plant in Arabidopsis.Besides,the fluorescence signal is quite strong at the newly-matured cell plate.Monitoring cytokinesis cells via FM4-64 dyeing,we found that PME 17 associates with the cell plate formation,expansion and maturation during the whole process of cytokinesis.2.Exocyst complex subunit SEC6 can regulate the localization of PME17 specificallyWe used SEC6 as a bait to screen Arabidopsis cDNA library via yeast two hybrid assay,and found that PME17 is an interacting protein with SEC6.Our lab has confirmed the interaction by yeast two-hybrid assay,BiFC(bimolecular fluorescence complementation)and pull-down.After crosstalk between PME17-GFP transgenic plant and PRsec6 mutant,we found that the localization of PME17 is disrupted without the presence of SEC6,the fluorescence signal is diffused in cytosol.To identify the regulation specificity of SEC6,we observe the localization pattern in different exocyst mutant background.Except the fluorescence signal is quite low in exo84b mutant,the pattern in those mutans does not change.These results suggest that SEC6 can regulate localization of PME17 specifically.3.Transport of PME17 goes through Golgi,TGN and PVC,but does not rely on clathrin-mediated endocytosis or cytoskeletonWe have also found punctate signals in cytoplasma while identifying the subcellular localization of PME17.To find out what are those punctaes,we crossed the transgenic plant with organelle markers.After observation of bifluorescence seedlings,PME17 can colocalize with Golgi,TGN(trans-Golgi network)and PVC(prevacuolar compartment),which indicates that transport of PME17 goes through Golgi,TGN.Through pharmaceutical treatment,we can further confirm the colocalization since PME17 is sensitive to BFA(Brefildin A),ConcA(Concanamycin A).Besides,PME17 is also sensitive to LY294002 and Wort(Wortmannin),which confirm the colocalization of PME17 and PVC,and this indicates that PME17 might transport to cell wall through PVC,or go to vacuole for degradation via PVC.However,PME17 is not sensitive to TyrA23(TyrphostinA23),CytD(Cytochalasin)and Oryzalin.The insensitivity suggests that PME17 trafficking does not rely on clathrin-mediated endocytosis or cytoskeleton.This study shows that PME17 transport to cell wall and cell plate through endomembrane system.However,PME17 cannot go outside of the cell in the absence of subunit SEC6,which indicate that SEC6 can recuit PME17 to the cell wall specifically.