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Specific accumulation of GFP in a non‐acidic vacuolar compartment via a C‐terminal propeptide‐mediated sorting pathway

Specific accumulation of GFP in a non‐acidic vacuolar compartment via a C‐terminal... The green fluorescent protein (GFP) from Aequorea victoria can be detected in living plant cells after transient transformation of protoplasts. Expression of the GFP can be used to monitor protein trafficking in a mixed cell population and also to study the different function and importance of organelles in different cell types. We developed a vacuolar form of GFP that was obtained by replacing the C‐terminal endoplasmic reticulum (ER)‐retention motif of mGFP5‐ER by the vacuolar targeting peptide of tobacco chitinase A. The vacuolar GFP was transported and accumulated in the vacuole as expected. However, we found two patterns of GFP accumulation after prolonged incubation (18–24 h) depending on the cell type. Most chloroplast‐rich protoplasts had a fluorescent large central vacuole. In contrast, most chloroplast‐poor protoplasts accumulated the GFP in one smaller vacuole but not in the large central vacuole, which was visible under a light microscope in the same cell. This differential accumulation reflected the existence of two different vacuolar compartments as described recently by immunolocalization of several vacuolar markers. We were able to characterize the vacuolar compartment to which GFP is specifically targeted as non‐acidic, since it did not accumulate neutral red while acidic vacuoles did not accumulate GFP. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Plant Journal Wiley

Specific accumulation of GFP in a non‐acidic vacuolar compartment via a C‐terminal propeptide‐mediated sorting pathway

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References (28)

Publisher
Wiley
Copyright
Copyright © 1998 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0960-7412
eISSN
1365-313X
DOI
10.1046/j.1365-313X.1998.00210.x
Publisher site
See Article on Publisher Site

Abstract

The green fluorescent protein (GFP) from Aequorea victoria can be detected in living plant cells after transient transformation of protoplasts. Expression of the GFP can be used to monitor protein trafficking in a mixed cell population and also to study the different function and importance of organelles in different cell types. We developed a vacuolar form of GFP that was obtained by replacing the C‐terminal endoplasmic reticulum (ER)‐retention motif of mGFP5‐ER by the vacuolar targeting peptide of tobacco chitinase A. The vacuolar GFP was transported and accumulated in the vacuole as expected. However, we found two patterns of GFP accumulation after prolonged incubation (18–24 h) depending on the cell type. Most chloroplast‐rich protoplasts had a fluorescent large central vacuole. In contrast, most chloroplast‐poor protoplasts accumulated the GFP in one smaller vacuole but not in the large central vacuole, which was visible under a light microscope in the same cell. This differential accumulation reflected the existence of two different vacuolar compartments as described recently by immunolocalization of several vacuolar markers. We were able to characterize the vacuolar compartment to which GFP is specifically targeted as non‐acidic, since it did not accumulate neutral red while acidic vacuoles did not accumulate GFP.

Journal

The Plant JournalWiley

Published: Aug 1, 1998

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