Evidence for nucleotide-dependent passive H⁺ transport protein in the plasma membrane of barley roots

Plasma membranes were isolated from barley roots by two-phase partitioning, and octylglucoside-soluble and -insoluble fractions were obtained. The insoluble fractions were reconstituted into liposomes, and the plasma membrane H⁺-ATPase was shown to participate in MgATP-dependent H⁺ transport activity. The H⁺ transport was decreased when the octylglucoside-soluble fraction was reconstituted together with the insoluble fraction. The decrease was not due to inhibition of the H⁺-ATPase, but rather was likely due to the increased H⁺ leakage from the proteoliposome. The octylglucoside-soluble fraction was, therefore, reconstituted in the liposomes and the passive H⁺ transport was determined using the pH jump method. A pH gradient across the membranes was generated by the pH jump, and the gradient was found to be dissipated by passive H⁺ transport. The H⁺ transport required ATP, K⁺, and valinomycin. The H⁺-transport also occurred when ADP, AMP, GTP, or ATP-γ-S was present instead of ATP, and did not occur when the octylglucoside-soluble fraction was boiled before the reconstitution. These findings suggest that nucleotide-dependent H⁺ transport protein is present in the plasma membrane of root cells.