Co-reconstitution of plasma membrane H(+)-ATPase from yeast and bacteriorhodopsin into liposomes. ATP hydrolysis as a function of external and internal pH.

The H(+)-ATPase from the plasma membrane of Saccharomyces cerevisiae was isolated and purified. The enzyme was reconstituted with bacteriorhodopsin into asolectin liposomes by detergent dialysis at a molar ratio of 1 H(+)-ATPase to 50 bacteriorhodopsins. The overall orientation of the proteins is such that proton pumping to the vesicle interior occurs upon illumination and after addition of ATP. All liposomes which contain H(+)-ATPase also contain bacteriorhodopsin. The rate of ATP hydrolysis was measured as function of pH in the dark and during illumination of the proteoliposomes. The pH dependency can be described by the protonation of a monovalent group from the outside with an apparent pK of 7.3 and the deprotonation of a monovalent group at the inside with an apparent pK of 3.7. Inside and outside refer to the orientation of the H(+)-ATPase in the liposomes which is opposite to that occurring in vivo. It is concluded that the first step in the reaction cycle is the binding of a proton from the cytosol which is followed by ATP binding, ATP hydrolysis on the enzyme and the release of ADP and phosphate, and finally the proton is released from the enzyme into the external medium.