Study On The Targeting Mechanism Of The Rice Vacuolar Phosphate Efflux Transporters

Phosphorus is one of the most important elements in plants,which account for 0.2%of plant dry weight.Because Phosphorus is a key component of many important biological macromolecules and involved in many physiological and biochemical progresses in plant,it's crucial for plant's growth and development.Vacuoles are the main Pi storage organelle in plants.Previous studies in our laboratory have identified two tonoplast localized glycerol-3-phosphate transporters(OsvGlpT1 and OsvGlpT2)in rice,which play a role in Pi effux into the cytoplasm from the vacuole.Glycerol-3-phosphate transporter was first found in E.coli,which was localized in the plasma membrane,while these two rice homologs reside on the tonoplast,and play different substrate characteristics.The purpose of this study was to analyze the tonoplast targeting mechanism of rice glycerol-3-phosphate transporters OsvGlpT1 and OsvGlpT2.The main findings of my research were list as follow:1.OsvGlpT1 and OsvGlpT2 targeted to the tonoplast through the ER-Golgi-TGN-PVC-Tonoplast.It showed that the dileucine motif in the loop between 6th and 7th transmembrane was the tonoplast sorting signal of these two proteins,replacing the dileucine motif by Ala residues(LL-AA)resulted in the modified protein mistargeting to plasma membrane;2.The COPII complex plays a crucial role in the process of exporting membrane proteins from ER to Golgi,We found that the subunit Sec24A of COPII complex could directly interact with OsvGlpTl via the diacidic motif E268K209D270 of OsvG1pT1.Mutation in the diacidic motif could abolish the interaction between OsvGlpTl with OsSec24A,and resulted in ER retention of OsvGlpTl.However,OsvGlpT2 could not directly interact with Os Sec24A,and its ER export signal is still obscue;3.OsPHF1 is essential for the ER export of OsPHT1 proteins,bimolecular fluorescence complementation experiments(BiFC)results showed that OsPHF1 could interact with OsvGlpT2 and OsSec24A,It suggested that OsPHFl may involved in the ER export process of OsvGlpT2.Together,these results showed that OsvGlpTl and OsvGlpT2 adopt different mechanisms for their ER export.Through directly interacting with OsSec24A,OsvGlpT1 was packaged into the COPII vesicle,and eventually reach the tonoplast.While OsvGlpT2 couldn't interact with OsSec24A,sorting of OsvGlpT2 into the COPII vesicle depends on OsPHF1 that physically link OsvGlpT2 with OsSec24A subunits,and eventually reach the tonoplast.We speculate that adopting different tonoplast targeting mechanisms may increase the fitness of plant to adapt to fluctuating external Pi availability by maintaining vacuole phosphate homestasis.