Journal Article

Characterization of Organelles in the Vacuolar-Sorting Pathway by Visualization with GFP in Tobacco BY-2 Cells

Naoto Mitsuhashi, Tomoo Shimada, Shoji Mano, Mikio Nishimura and Ikuko Hara-Nishimura

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 41, issue 9, pages 993-1001
Published in print September 2000 | ISSN: 0032-0781
Published online September 2000 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcd040
Characterization of Organelles in the Vacuolar-Sorting Pathway by Visualization with GFP in Tobacco BY-2 Cells

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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.

Keywords: Key words: BY-2 cells — Endoplasmic reticulum — GFP — Golgi complex — Vacuolar-sorting receptor — Vacuole.; Abbreviations: BCECF, 2,7-bis-(2-carboxyethyl)-5,(6)-carboxyfluorescein; CTPPs, C-terminal propeptides; DEX, dexamethasone; EGF, an epidermal growth factor; ER, endoplasmic reticulum; EST, expressed sequence tag; GFP, green fluorescent protein; NTPPs, N-terminal propeptides; PV72, a vacuolar-sorting receptor of pumpkin; TIP, tonoplast intrinsic protein.

Journal Article.  6207 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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