Dephosphorylation activates the purified plant plasma membrane H+-ATPase--possible function of phosphothreonine residues in a mechanism not involving 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 separation on a glycerol gradient, a fraction with a high specific activity of 9 micromol ATP x min(-1) x mg protein(-1) was obtained, in which the H+-ATPase polypeptide represented at least 80% of the protein. The incubation of this fraction in the presence of alkaline phosphatase increased H+-ATPase activity by 40%, in a manner consistent with dephosphorylation of the enzyme itself. The hydrolytic activity of the solubilized enzyme and its proton translocating activity, after reconstitution into proteoliposomes, were stimulated to the same extent. Alkaline phosphatase treatment was also accompanied by a 92% decrease in the H+-ATPase 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 activated by lysophosphatidylcholine addition nor by trypsin-mediated proteolysis, 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 phosphorylation evoked by alkaline phosphatase most likely differs from the autoinhibitory control of the H+-ATPase by its C-terminal domain.
Desbrosses
G
G
Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique (URA 2133) et Ecole Nationale Supérieure d'Agronomie, Montpellier, France.
Stelling
J
J
Renaudin
J P
JP
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D008244:Lysophosphatidylcholines / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D010947:Plants, Toxic; D006180:Proton-Translocating ATPases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology
D008244:Lysophosphatidylcholines; D010769:Phosphothreonine; D000469:Alkaline Phosphatase; D014357:Trypsin; D006180:Proton-Translocating ATPases