Trafficking of phosphatidylinositol 3-phosphate from the trans-Golgi network to the lumen of the central vacuole in plant cells

Very limited information is available on the role of phosphatidytinositol 3 -phosphate (P1[3]P) in vesicle trafficking in plant cells. To investigate t he role of PI(3)P during the vesicle trafficking in plant cells, we exploit ed the Pt(3)P-specific binding property of the endosome binding domain (EBD ) (amino acids 1257 to 1411) of human early endosome antigen 1, which is in volved in endosome fusion. When expressed transiently in Arabidopsis protop lasts, a green fluorescent protein (GFP):EBD fusion protein exhibited PI(3) P-dependent localization to various compartments-such as the trans-Golgi ne twork, the prevacuolar compartment, the tonoplasts, and the vesicles in the vacuolar lumen-that varied with time. The internalized GFP:EBD eventually disappeared from the lumen. Deletion experiments revealed that the P1(3)P-d ependent localization required the Rab5 binding motif in addition to the zi nc finger motif. Overexpression of GFP:EBD inhibited vacuolar trafficking o f sporamin but not trafficking of Hf-ATPase to the plasma membrane. On the basis of these results, we propose that the trafficking of GFP:EBD reflects that of P1(3)P and that P1(3)P synthesized at the trans-Golgi network is t ransported to the vacuole through the prevacuolar compartment for degradati on in plant cells.