Dimer formation of proteolipid constructs. (A) Mesophyll protoplasts of A. thaliana were co-transfected with proteolipid subunits fused to CFP and YFP, respectively. A fusion protein of the transmembrane segments 3–4 of VHA-c and helices 3–4 of VHA-c"1 was engineered and is refered to as hybrid proteolipid. Observation of an energy transfer > 10% is taken as tentative indicator for the presence of more than one copy of the examined subunit in the complex. The data represent the mean average ± SE (VHA-c: n = 80; VHA-c": n = 71; hybrid proteolipid: n = 121; VHA-c"&VHA-c"trunc: n = 59; VHA-e1: n = 85; VHA-e2: n = 50; VHA-e1&VHA-e2: n = 78). (B) Bimolecular fluorescence complementation approach using VHA-c"-YFP-N-terminus and VHA-c"-YFP-C-terminus (VHA-c", n = 53). The BiFC-constructs and 35S-CFP-NosT were cotransfected. The YFP-fluorescence was normalised to CFP-fluorescence. YFP-formation of fluorophore fragments driven by the CaMV35S-promoter served as control for cytosolic self-assembly of the fluorophore (split YFP, n = 61) and expression of CFP under control of the CaMV35S-promoter served as background control (CFP, n = 25). The data represent the mean average ± SE.
Seidel et al. BMC Cell Biology 2008 9:28 doi:10.1186/1471-2121-9-28