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Mapping of C‐termini of V‐ATPase subunits by in vivo‐FRET measurements
Author(s) -
Seidel Thorsten,
Golldack Dortje,
Dietz Karl-Josef
Publication year - 2005
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2005.06.077
Subject(s) - förster resonance energy transfer , yellow fluorescent protein , endomembrane system , chemistry , biophysics , green fluorescent protein , v atpase , protein subunit , fluorescence , in vivo , vacuole , atpase , microbiology and biotechnology , biochemistry , membrane , biology , cytoplasm , enzyme , vesicle , physics , quantum mechanics , gene
The plant V‐ATPase is a protein complex of 13 different VHA‐subunits and functions as ATP driven motor that electrogenically translocates H + into endomembrane compartments. The central rotor extends into the hexameric head that is fixed by peripheral stators to an eccentric membrane domain. The localization and orientation of VHA‐subunits of the head and peripheral stalk region were investigated by in vivo fluorescence resonance energy transfer (FRET). To this end, VHA‐E, VHA‐G, VHA‐H of the peripheral stalks as well as subunits VHA‐A and VHA‐B were C‐terminally fused to cyan (CFP) and yellow fluorescent protein (YFP). Protoplasts transfected with FRET‐pairs of CFP‐donor and YFP‐acceptor fluorophores fused to VHA‐subunits were analysed for FRET by laser scanning microscopy. The result of the C‐termini mapping allows to refine the arrangement and interaction of the subunits within the V‐ATPase complex in vivo. Furthermore, expression of fused VHA‐E and VHA‐H stimulated acidification of protoplast vacuoles, while other constructs had no major effect on vacuolar pH tentatively indicating a regulatory role of these subunits in plants.