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Fig. 6. Subcellular localization of A-GFP mutated at specific residues within region n. The indicated mutants (see text for details) were expressed in MDCK cells. Twenty-four hours after transfection, cells were fixed and stained with the CTR433 monoclonal Golgi marker. The subcellular localization of the mutants was examined by conventional fluorescence microscopy. For each fusion protein, GFP fluorescence and CTR433 immunofluorescence images are shown, with the corresponding overlay image (GFP, green; CTR433, red) in the right-hand column. When compared to the wild-type A-GFP (top panels), mutation of charged residues within region n (AYKEKMKEL) to either alanine (KKK/AAA; EE/AA) or to conservatively charged amino acids (KKK/RRR; EE/DD) disturbed the subcellular localization of the resulting fusion proteins. In contrast to the wild type, which is only localized to the Golgi and punctate structures, these mutants were distributed both at the Golgi complex and at the plasma membrane, as clearly observed at cell junctions. Bar, 10 µm.
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