Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells.

摘要: Abstract The proton-translocating plant vacuolar H+-ATPase (VHA) is of prime importance for acidification intracellular compartments and essential processes such as secondary activated transport, maintenance ion homeostasis, adaptation to environmental stress. Twelve genes have been identified that encode subunits the functional V-ATPase complex. In this study, c a from Mesembryanthemum crystallinum were fused cyan fluorescent protein (CFP) yellow (YFP), respectively, transiently coexpressed in protoplasts. Two-colour scanning confocal fluorescence microscopy demonstrates fusion proteins VHA-c-CFP VHA-a-YFP are colocalized at tonoplast, plasmamembrane, endoplasmic membrane structures indicating expression cytoplasmic vesicles. Furthermore, resonance energy transfer (FRET) was used visualize interaction VHA-c VHA-a vivo on nanometer length scale. Excitation CFP donor fluorophore caused increased emission YFP-fluorescence protoplasts due FRET. Our results give strong evidence physical living cells.