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 phospholip ids (phosphatidylcholine), with removal of detergent by passage throug h an Amberlite XAD-2 column. The specific ATP-hydrolysis activity of t he proteoliposomes was increased 4-fold over the activity of the nativ e tonoplast vesicles. Pumping of protons by the reconstituted ATPase w as demonstrated by quinacrine-fluorescence quenching. Neither vanadate nor azide inhibited ATP-hydrolysis and transport activity. Both the A TP-hydrolysis and the H+-translocating activities were inhibited by ni trate, the first activity being the more sensitive one. Both activitie s were completely inhibited by bafilomycin A(1), an inhibitor of vacuo lar 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 cat ions. The proteoliposomes showed no PP(i)ase activity. SDS-PAGE showed that the reconstituted H+-ATPase from Citrus contained polypeptides o f apparent molecular masses of 63, 52, 37, 33 and 16 kDa that cross-re acted with an antiserum against the tonoplast ATPase from Kalanchoe da igremontiana. Freeze-fracture electron microscopy of the proteoliposom es showed that proteins were incorporated into the bilayer membrane. T he proteoliposomes from Citrus were mainly vesicles intramembraneous w ith particles whose diameter ranged from 6 to 7 nm. The results conclu sively demonstrate a selective reconstitution of a functional H+-ATPas e from Citrus.