Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells.
The proton-translocating plant vacuolar H(+)-ATPase (VHA) is of prime importance for acidification of intracellular compartments and is essential for processes such as secondary activated transport, maintenance of ion homeostasis, and adaptation to environmental stress. Twelve genes have been identified that encode subunits of the functional V-ATPase complex. In this study, subunits c and a of the V-ATPase from the plant Mesembryanthemum crystallinum were fused to cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, and were transiently coexpressed in protoplasts. Two-colour scanning confocal fluorescence microscopy demonstrates that the fusion proteins VHA-c-CFP and VHA-a-YFP are colocalized at the tonoplast, the plasmamembrane, and at endoplasmic membrane structures indicating expression in cytoplasmic vesicles. Furthermore, fluorescence resonance energy transfer (FRET) was used to visualize the interaction of VHA-c and VHA-a in vivo on the nanometer length scale. Excitation of CFP as donor fluorophore caused increased emission of YFP-fluorescence in protoplasts due to FRET. Our results give strong evidence for physical interaction of subunits c and a in living plant cells.
Department of Physiology and Biochemistry of Plants, W5, University of Bielefeld, 33501 Bielefeld, Germany.
Thorsten
T
Seidel
Christoph
C
Kluge
Miriam
M
Hanitzsch
Joachim
J
Ross
Markus
M
Sauer
Karl-Josef
KJ
Dietz
Dortje
D
Golldack
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D035723:Mesembryanthemum / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011523:Protoplasts / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*
D010940:Plant Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006180:Proton-Translocating ATPases