PROPERTIES OF PROTON AND SUGAR-TRANSPORT AT THE TONOPLAST OF TOMATO (LYCOPERSICON-ESCULENTUM) FRUIT

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A(1), but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF brit was insensitive to nitrate, bafilomycin A(t), vanadate and azide. Kinetic studies of PPase activity gave an apparent K-m for PPi of 18 mu M. Identical distributions of bafilomycin- and NO3--sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin-sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35-day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP- and PPi-dependent Delta pH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H+ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H+/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PPi) stimulation of [C-14]-sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy-requiring uptake mechanisms at the tonoplast.