ATP synthesis by the F₀F₁ ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin: 1. Factors defining the optimal reconstitution of ATP synthases with bacteriorhodopsin

Optimal conditions for the reconstitution of bacteriorhodopsin and H⁺-transporting ATP synthase from thermophilic Bacillus PS3 (TF₀F₁) were determined. Phosphatidylcholine/phosphatidic acid liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, octaethylene glycol n-dodecylether, sodium cholate or sodium deoxycholate and the incorporation of proteins by these detergents was studied at each step of the solubilization process. After removal of detergent by means of SM-2 Bio-Beads, the light-driven ATP synthase activities of the resulting proteoliposomes were analyzed at 40°C. The nature of the detergent used for reconstitution was important for determining the mechanism of protein insertions. The most efficient reconstitutions were obtained with octyl glucoside or Triton X-100 by insertion of the proteins into detergent-saturated liposomes. The conditions for reconstitution were further optimized with regard to functional coupling between bacteriorhodopsin and TF₀F₁. It was demonstrated that one of the main factors limiting the production of efficient reconstituted proteoliposomes was related to activation of the highly stable TF₀F₁. Activation was accomplished by total solubilization of phospholipids and proteins in a Triton X-100/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold stimulation of the ATP synthase activity. Final ATP synthase activities depended greatly on the lipid/bacteriorhodopsin and the lipid/TF₀F₁ ratios as well as on the phospholipid used. In particular, light-driven ATP synthesis depended upon the presence of negatively charged phospholipids. Cholesterol was found to induce a fourfold increase in ATP synthase activity with a concomitant 65% decrease in the K_m for ADP, suggesting that sterols can modulate catalytic events mediated by F₁. Preparations obtained by this step-by-step reconstitution procedure displayed activities up to 20-fold higher (500-800 nmol ATP · min^-1 · mg TF₀F₁ ^-1 in the presence of cholesterol) than the maximal values reported in the literature for light-driven ATP synthesis by TF₀F₁ measured under similar conditions. This study also allowed rationalization of the different parameters involved in reconstitution experiments and the present simple method is shown to be of general use for preparation of efficient proteoliposomes containing bacteriorhodopsin and choloroplast or mitochondrial F₀F₁-type ATP synthases.