Subcellular localization of AtOCT1-AtOCT6 using cDNA-GFP fusions and protoplast transformation. Left part: Subcellular localisation studies of AtOCT-GFP fusion proteins. OCT proteins were fused with GFP at the C-terminus and expressed transitionally in protoplasts of Arabidopsis Col-0 cell cultures. Protoplasts expressing the GFP protein under the control of the CaMV 35S promoter were used as control. GFP fluorescence was detected throughout the cytosol. The GFP fluorescence (left panel) and the bright field image (middle panel) were overlayed in the right panel. AtOCT1-GFP fluorescence was detected at the plasma membrane as shown before . Fusion proteins of AtOCT2-AtOCT6-GFP were localized at membranes in the protoplasts, most probably at the tonoplast. Right part: To confirm the assumed vacuolar localisation, vacuoles were released by an osmotic shock from the protoplasts. GFP signal of the fusion proteins of AtOCT2-AtOCT6-GFP is detected at the free vacuole or at vacuoles just escaping from the protoplasts. Free GFP signal is localised to the cytosol.