Characterization of organelles in the vacuolar-sorting pathway by visualization with GFP in tobacco BY-2 cells

We have shown the localization and mobilization of modified green fluoresce nt proteins (GFPs) with various signals in different compartments in a vacu olar-sorting system of tobacco BY-2 cells. In contrast to the efficient sec retion of GFP from the transformed cells expressing SP-GFP composed of a si gnal peptide and GFP, accumulation of GFP in the vacuoles was observed in t he cells expressing SP-GFP fused with the C-terminal peptide of pumpkin 2S albumin. This indicated that this peptide is sufficient for vacuolar target ing, Interestingly, the fluorescence in the vacuoles disappeared sharply at 7 d after inoculation of the cells, but it appeared again after re-inocula tion into a new culture medium. When SP-GFP was fused with the region, term ed PV72C, including a transmembrane domain and a cytosolic tail of a vacuol ar-sorting receptor PV72, GFP-PV72C was detected in the Golgi-complex-like small particles. Prolonged culture showed that GFP-PV72C that reached the p revacuolar compartments was cleaved off the PV72C region to produce GFP, th at arrived at the vacuoles to be diffused. These findings suggested that th e vacuolar-sorting receptor might be recycled between the Golgi complex and prevacuolar compartments.