Characterization of two new phosphorylation sites involved in the regulation of plant plasma membrane H+-ATPases

(eng) The plant plasma membrane H+-ATPase couples ATP hydrolysis to the transport of protons out of the cell, thus generating an electrochemical gradient used by secondary transporters to move ions and metabolites through the membrane. In addition, H+-ATPase is involved in several physiological roles such as stomata opening and cell elongation. This enzyme is highly regulated by (de)phosphorylation of several residues and binding of regulatory proteins. A well characterized example is the enzyme activation by phosphorylation of its penultimate residue, a Thr, and the subsequent binding of 14-3-3 regulatory proteins. Mass spectroscopy analysis of purified PMA4 and PMA2, the most broadly expressed Nicotiana plumbaginifolia plasma membrane H+-ATPases, led to the identification of new phosphorylated residues. Two of them were studied in more details. PMA4 Ser448 and PMA2 Thr889 were both mutated into aspartate or alanine and the mutant H+-ATPases were expressed in the yeast Saccharomyces cerevisae as well as in Nicotiana tabacum suspension cells. Both mutations of PMA4 Ser448 resulted, in BY2 cells as in yeast, in higher penultilmate residue phosphorylation than in wild-type. However, unlike in yeast, the ATPase activity was lower in BY2 cells expressing the PMA4 Ser448 mutants compared to that expressing wild–type PMA4. The PMA4-S448D mutant conferred higher proton pumping activity than the wild–type PMA4 and the PMA4-S448A mutant, suggesting that the former has a better H+ pumping/ATP hydrolysis coupling. Concerning PMA2, the PMA2-T889A mutation did not allow yeast growth while PMA2-T889D resulted in improved growth and increased H+-ATPase activity in spite of reduced PMA2 penultimate Thr phosphorylation and 14-3-3 protein binding. Combining PMA2-T889D with other mutations indicated that PMA2 Thr889 phosphorylation can activate the H+-ATPase, provided that PMA2 penultimate residue is phosphorylated. Interestingly, 14-3-3 protein binding was not required but brought additional activation. Expression of these mutants in BY2 cells led to similar observations. In conclusion, this work highlighted different regulatory features of two newly identified phosphorylation sites of plant H+-ATPases. In particular, they show that this enzyme can be activated in the absence of activating 14-3-3 proteins.