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A simple method for discriminating between cell membrane and cytosolic proteins
Author(s) -
Serna Laura
Publication year - 2005
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/j.1469-8137.2004.01278.x
Subject(s) - green fluorescent protein , cytosol , protein subcellular localization prediction , microbiology and biotechnology , cytoplasm , biology , plasmolysis , subcellular localization , fluorescence recovery after photobleaching , cell membrane , cell , membrane protein , fusion protein , organelle , biochemistry , membrane , cell wall , gene , recombinant dna , enzyme
Summary• Transgenic plants expressing either green fluorescent protein (GFP)‐genomic DNA or GFP‐cDNA fusions have been used as powerful tools to define the subcellular localization of many proteins. Because most plant cells are highly vacuolated, the cytosol is confined to a thin layer at the periphery of the cells, making it very difficult to distinguish among cell wall, cell membrane and cytosolic GFP‐fusion proteins. • Plasmolysis tests inform about cell‐wall localization of GFP‐tagged proteins, but they do not discriminate between its cell membrane and/or cytoplasmic localization. By observing the GFP signal in transgenic protoplasts placed at a hypotonic solution, it was possible to distinguish between cell membrane and cytosolic GFP‐tagged proteins. • The osmotic disruption of the protoplast vacuole in the hypotonic solution allows the diffusion of the GFP signal from the cell periphery to the central part of the cell volume when the GFP is fused to a soluble protein. By contrast, such diffusion does not occur when the protein under study is attached to the cell membrane. • The present method is easier, faster and cheaper than subcellular fractionating studies and/or immunoelectron microscopy, which have been traditionally used to discern between cell membrane and cytosolic proteins.