Plant and Cell Physiology, 2000, Vol. 41, No. 9 993-1001
© 2000 Oxford University Press
Paper |
Characterization of Organelles in the Vacuolar-Sorting Pathway by Visualization with GFP in Tobacco BY-2 Cells
1 Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan 2 Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Okazaki, 444-8585 Japan 3 Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606-8502 Japan
Abstract
We have shown the localization and mobilization of modified green fluorescent proteins (GFPs) with various signals in different compartments in a vacuolar-sorting system of tobacco BY-2 cells. In contrast to the efficient secretion of GFP from the transformed cells expressing SP-GFP composed of a signal peptide and GFP, accumulation of GFP in the vacuoles was observed in the cells expressing SP-GFP fused with the C-terminal peptide of pumpkin 2S albumin. This indicated that this peptide is sufficient for vacuolar targeting. Interestingly, the fluorescence in the vacuoles disappeared sharply at 7 d after inoculation of the cells, but it appeared again after re-inoculation into a new culture medium. When SP-GFP was fused with the region, termed PV72C, including a transmembrane domain and a cytosolic tail of a vacuolar-sorting receptor PV72, GFP-PV72C was detected in the Golgi-complex-like small particles. Prolonged culture showed that GFP-PV72C that reached the prevacuolar compartments was cleaved off the PV72C region to produce GFP, that arrived at the vacuoles to be diffused. These findings suggested that the vacuolar-sorting receptor might be recycled between the Golgi complex and prevacuolar compartments.
Footnotes
4 Corresponding author: E-mail, ihnishi@gr.bot.kyoto-u.ac.jp; Fax, +81-75-753-4141.
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