The V-ATPase (VHA) is a protein complex of 13 different VHA-subunits. It functions as an ATP driven rotary-motor that electrogenically translocates H+ into endomembrane compartments. In Arabidopsis thaliana V-ATPase is encoded by 23 genes posing the question of specific versus redundant function of multigene encoded isoforms.The transmembrane topology and stoichiometry of the proteolipid VHA-c" as well as the stoichiometry of the membrane integral subunit VHA-e within the V-ATPase complex were investigated by in vivo fluorescence resonance energy transfer (FRET). VHA-c", VHA-e1 and VHA-e2, VHA-a, VHA-c3, truncated variants of VHA-c3 and a chimeric VHA-c/VHA-c" hybrid were fused to cyan (CFP) and yellow fluorescent protein (YFP), respectively. The constructs were employed for transfection experiments with Arabidopsis thaliana mesophyll protoplasts. Subcellular localization and FRET analysis by confocal laser scanning microscopy (CLSM) demonstrated that (i.) the N- and C-termini of VHA-c" are localised in the vacuolar lumen, (ii.) one copy of VHA-c" is present within the VHA-complex, and (iii.) VHA-c" is localised at the ER and associated Golgi bodies. (iv.) A similar localisation was observed for VHA-e2, whereas (v.) the subcellular localisation of VHA-e1 indicated the trans Golgi network (TGN)-specifity of this subunit.The plant proteolipid ring is a highly flexible protein subcomplex, tolerating the incorporation of truncated and hybrid proteolipid subunits, respectively. Whereas the membrane integral subunit VHA-e is present in two copies within the complex, the proteolipid subunit VHA-c" takes part in complex formation with only one copy. However, neither VHA-c" isoform 1 nor any of the two VHA-e isoforms were identified at the tonoplast. This suggest a function in endomembrane specific VHA-assembly or targeting rather than proton transport.