The plasma-membrane H+-ATPase from beet root is inhibited by a calcium-dependent phosphorylation

Several plasma-membrane proteins from beet root (Beta vulgaris L.) have been functionally incorporated into reconstituted proteoliposomes. These showed H⁺-ATPase activity, measured both as ATP hydrolysis and H⁺ transport. The proton-transport specific activity was 10 times higher than in plasma membranes, and was greatly stimulated by potassium and valinomycin. These proteoliposomes also showed calcium-regulated protein kinase activity. This kinase activity is probably due to a calmodulin-like domain protein kinase (CDPK), since two protein bands were recognized by antibodies against soybean and Arabidopsis CDPK. This kinase phosphorylated histone and syntide-2 in a Ca²⁺-dependent manner. Among the plasma-membrane proteins phosphorylated by this kinase, was the H⁺-ATPase. When the H⁺-ATPase was either prephosphorylated or assayed in the presence of Ca²⁺, both the ATP-hydrolysis and the proton-transport activities were slower. This inhibition was reversed by an alkaline-phosphatase treatment. A trypsin treatment (that has been reported to remove the C-terminal autoinhibitory domain from the H⁺-ATPase) also reversed the inhibition caused by phosphorylation. These results indicate that a Ca²⁺-dependent phosphorylation, probably caused by a CDPK, inhibits the H⁺-ATPase activities. The substrate of this regulatory phosphorylation could be the H⁺-ATPase itself, or a different protein influencing the ATPase activities. Received: 1 May 1997 / Accepted: 25 June 1997