Mapping of C-termini of V-ATPase subunits by in vivo-FRET measurements.
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.
Seidel
Thorsten
T
Biochemistry and Physiology of Plants, W5, University of Bielefeld, Universitaetsstrasse 25, D-33501 Bielefeld, Germany.
Golldack
Dortje
D
Dietz
Karl-Josef
KJ
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011523:Protoplasts / Q000737:chemistry / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D025262:Vacuolar Proton-Translocating ATPases