SOLUBILIZATION AND FUNCTIONAL RECONSTITUTION OF THE TONOPLAST H+-ATPASE FROM CITRUS IN LIPOSOMES

Proteoliposomes were formed after solubilization of tonoplast proteins from Citrus with octylglucoside in the presence of soybean phospholipids (phosphatidylcholine), with removal of detergent by passage through an Amberlite XAD-2 column. The specific ATP-hydrolysis activity of the proteoliposomes was increased 4-fold over the activity of the native tonoplast vesicles. Pumping of protons by the reconstituted ATPase was demonstrated by quinacrine-fluorescence quenching. Neither vanadate nor azide inhibited ATP-hydrolysis and transport activity. Both the ATP-hydrolysis and the H+-translocating activities were inhibited by nitrate, the first activity being the more sensitive one. Both activities were completely inhibited by bafilomycin A(1), an inhibitor of vacuolar type ATPase, at concentrations of 20 nM. A potent inhibitor of H+-transport, DCCD, completely inhibited both activities at 100 mu M and with half-maximal inhibition at concentrations of 40 mu M and 60 mu M for the H+-transport and the ATP-hydrolysis activities, respectively. The reconstituted H+-ATPase was stimulated by anions (Cl- > Br- > SO42-), inhibited by NO3- and I- and largely insensitive to monovalent cations. The proteoliposomes showed no PP(i)ase activity. SDS-PAGE showed that the reconstituted H+-ATPase from Citrus contained polypeptides of apparent molecular masses of 63, 52, 37, 33 and 16 kDa that cross-reacted with an antiserum against the tonoplast ATPase from Kalanchoe daigremontiana. Freeze-fracture electron microscopy of the proteoliposomes showed that proteins were incorporated into the bilayer membrane. The proteoliposomes from Citrus were mainly vesicles intramembraneous with particles whose diameter ranged from 6 to 7 nm. The results conclusively demonstrate a selective reconstitution of a functional H+-ATPase from Citrus.