DEPHOSPHORYLATION ACTIVATES THE PURIFIED PLANT PLASMA-MEMBRANE H-ATPASE - POSSIBLE FUNCTION OF PHOSPHOTHREONINE RESIDUES IN A MECHANISM NOTINVOLVING THE REGULATORY C-TERMINAL DOMAIN OF THE ENZYME()

The plasma membrane H+-ATPase was purified from tobacco cells (line BY -2). After solubilization by lysophosphatidylcholine followed by separ ation on a glycerol gradient, a fraction with a high specific activity of 9 mu mol ATP . min(-1). mg protein(-1) was obtained, in which the H+-ATPase polypeptide represented at least 80% of the protein. The inc ubation of this fraction in the presence of alkaline phosphatase incre ased H+-ATPase activity by 40%, in a manner consistent with dephosphor ylation of the enzyme itself. The hydrolytic activity of the solubiliz ed enzyme and its proton translocating activity, after reconstitution into proteoliposomes, were stimulated to the same extent. Alkaline pho sphatase treatment was also accompanied by a 92% decrease in the H+-AT Pase phosphothreonine content, whereas the phosphoserine residues were almost unaffected. The dephosphorylation induced a slight decrease of the affinity of the enzyme towards ATP. The purified enzyme was not a ctivated by lysophosphatidylcholine addition nor by trypsin-mediated p roteolysis, two treatments reported to release the inhibitory control by the C-terminal domain of the H+-ATPase and to increase the affinity of the enzyme towards ATP. Based on these results, the regulatory pho sphorylation evoked by alkaline phosphatase most likely differs from t he autoinhibitory control of the H+-ATPase by its C-terminal domain.