Exocyst Subunit EXO70A1 Plays A Unique Role In The Localization Of Patellin3 To The Plasma Membrane And The Cell Plate

Protein trafficking is essential for the plasma membrane dynamics and cytokinesis of plant cells.In the endomembrane system,membrane trafficking pathways allow the delivery of cellular proteins to their site of action via exocytosis and endocytosis.The exocyst,an evolutionarily conserved octameric vesicle tethering complex,functions upstream of SNARE(soluble NSF attachment protein receptor)mediated vesicle fusion with the plasma membrane.Previous studies showed that the exocyst complex is involved in multiple functions in plant cells,including cell division,cell polarity and morphogenesis,cell wall biogenesis,integral plasma membrane proteins recycling and polarization,plant immune response,autophagy and so on.In Arabidopsis,EXO70A1,a subunit of the exocyst complex,is one of the most abundant isoform in the 23 EXO70 family proteins and is known to be involved in cytokinesis,cell polarity and morphogenesis,seed coat biogenesis and PINs recycling.That indicated EXO70A1 may functions as a member of the Exocyst complex.From our research,we gained a new understanding that EXO70A1 functions.1.Identification of PATL3 as a EXO70A1 interacting proteinA yeast two-hybrid(Y2H)screen of an Arabidopsis cDNA library was performed using full-length EXO70A1 as bait and an interacting protein Patellin3(PATL3)was identified.Patellin family has six members named PATL1-6,each of them contains a phospholipid binding domain(Sec 14 domain)and a protein-protein interaction domain(Gold domain).Subsequently,a series of experiments were performed to verify the Y2H results.GST Pull-down assays indicated a direct interaction between PATL3 and EXO70A1 in vitro,while co-immunoprecipitation(Co-IP)and luciferase complementation imaging(LCI)assays suggested that they also interact with each other in vivo,and bimolecular fluorescence complementation(BiFC)data showed that PATL3 associates with EXO70A1 at the site of the plasma membrane in plant cells.Segmentation analysis of PATL3 revealed that the C-terminal GOLD domain of PATL3 was required for the interaction.2.PATL3 is mainly located in the cytoplasm and plasma membrane in interphase cells,while it is mainly located in the cell plate and plasma membrane during cytokinesisTransgenic Arabidopsis plants and tobacco BY-2 cells stably expressing the 35S promoter driven PATL3-GFP(green fluorescent protein)were observed by confocal microscope,we found that PATL3 is mainly located in the cytoplasm and plasma membrane in interphase cells,while it is mainly located in the cell plate and plasma membrane during cytokinesis.Using confocal microscope and in conjunction with the styryl dye FM4-64,we followed the distribution of fluorescent signals in a single dividing cell and found that PATL3 presents at the cell plate during the whole process of cytokinesis associated with the cell plate formation,expansion and maturation.In the transgenic Arabidopsis plants of proPATL3:PATL3-GFP,PATL3 showed the same location as the over-expression lines.3.The localization of PATL3 to the plasma membrane and the cell plate is dependent on EXO70A1To explore the significance of the interaction between EXO70A1 and PATL3,we obtained pro35S:PATL3-GFP and pro35S:EXO70A1-mCherry double fluorescence labeled Arabidopsis lines by hybridization,on the other hand,we also obtained the exo70al mutants expressing pro35S:PATL3-GFP.In the double fluorescence transgenic plants,PATL3-GFP colocalizes with EXO70A1-mCherry at the plasma membrane and the cell plate.In the exo70al homozygous mutants expressing PATL3-GFP,PATL3 could not reach the the plasma membrane and the cell plate anymore,but accumulated as dots inside the cytosol,indicated that EXO70A1 is required for the localization of PATL3 to the plasma membrane and the cell plate.These dots are insensitive to BFA,and do not co-localize with markers of endomembrane compartments tested.These data suggest that PATL3 to the plasma membrane and the cell plate is not via the endomembrane system.Bioinformatics data also show that the PATL3 protein sequence does not contain a signal peptide and transmembrane sequence.Additionally,P ATL3-GFP fluorescence intensity was significantly decreased.RT-PCR results indicated that is not because of gene transcription as the transcript level of PATL3 is essentially unchanged.However,the content of PATL3 protein was significantly decreased in exo70al mutants.These results indicated that the localization of PATL3 to the plasma membrane and the cell plate is dependent on EXO70A1 and if can not correctly positioned,it will be degraded.4.The membrane localization of PATL3 is regulated by lipid and is independent on cytoskeleton/EXPO/SEC6/EX084bPATL3 is affected by the specific PI3K inhibitor wortmannin(Wort),but differed from that of the Wort-induced enlargement and vacuolation of PVCs,Wort caused diffusion of these dot fluorescent signals.These results suggested that PATL3 could combine PI(3)P and its derived phospholipids in the cytosol.Protein-lipid overlay assays confirmed that PATL3 was able to bind PI(3)P and its derived phospholipids,which indicated that PATL3 could bind lipids in the cytosol and the plasma membrane.After being recruited by EXO70A1 via Gold domain to the membranes,PATL3 might be able to bind PI(3)P and its derived phospholipids on the menbranes.The PI3K inhibitors(Wort and LY294002)strongly inhibit the plasma membrane and cell plate localization of PATL3 confirms this hypothesis.PATL3 is unaffected by actin depolymerizing drugs CytD and microtubule depolymerization drug Oryzalin,suggested that the trafficking of PATL3 was independent on the cytoskeleton.A novel exocyst-positive organelle named EXPO,defined by Exocyst subunit EXO70E2,is not affected by BFA,ConcA and Wort,which can mediate the process of non conventional protein secretion in Arabidopsis thaliana.PATL3 shows no difference in exo70e2 mutants,suggested that the membrane localization PATL3 is independent on EXPO.In addition,PATL3-GFP in sec6 and exo84b mutants also do not change,suggested that the membrane localization of PATL3 was also independent of SEC6 and EX084b,and it also suggested that EXO70A1 plays a specific role on the targeting of PATL3 to the membranes.5-The membrane localization of all Patellins maybe dependent on EXO70A1Microarray data indicated that the Patellins were ubiquitously expressed in all plant organs as well as at different stages of plant development w:ith a high expression level except for PATL5.Promoter activity analysis further confirmed that PATL1-4 and PATL6 are ubiquitously expressed.This implies that their function may be redundant.Localization analyses revealed that PATL1,2,4,6-GFP showed the same location as PATL3.In addition,BiFC assays showed that PATL1 can also interact with EXO70A1 at the site of the plasma membrane.Furthermore,EXO70A1 can interact with all Gold domain of Patellin family.These data suggested that their membrane localization maybe dependent on EXO70A1.Taken together,we conclude that Exo70A1 recruits PATLs directly to the membrane,which is independent on its role in secretory/recycling vesicle tethering.