Characterization of the protein phosphatase regulating the plasma membrane H⁺-ATPase

Abstract

Plant plasma membrane H⁺-ATPase creates an electrochemical gradient of H⁺ across the plasma membrane. Recent studies have demonstrated that the H⁺-ATPase is activated via phosphorylation on a penultimate threonine residue in the C-terminus and subsequent binding of 14-3-3 protein to the phosphorylated C-terminus. However, the biochemical properties of protein kinase and phosphatase for the H⁺-ATPase are largely unknown.
In this study, we investigated in vitro dephosphorylation of the H⁺-ATPase. Dephosphorylation of the H⁺-ATPase was detected in the plasma membrane from etiolated seedlings of Arabidopsis. Furthermore, the dephosphorylation was specifically inhibited by EDTA a chelating agent for divalent cations, but not by calyculin A, an inhibitor of type 1 and type 2A protein phosphatases. Moreover, we found that dephosphorylation required Mg²⁺ or Mn²⁺. These results indicate that type 2C protein phosphatase is likely to be involved in dephosphorylation. We will report the proteomic analysis of the 1% Triton X-100-insoluble fraction that possesses dephosphorylation activity for the H⁺-ATPase.