In vitro phosphorylation and dephosphorylation of the plasma membrane H⁺-ATPase from Vicia guard cells.

Abstract

Blue light induces stomatal opening through the activation of the plasma membrane H⁺-ATPase. Recent investigations have demonstrated that blue light activates the H⁺-ATPase via phosphorylation on threonine residue in the C-terminus with subsequent binding of 14-3-3 protein to the phosphorylated C-terminus in guard cells. However, protein kinase and protein phosphatase, which catalyze phosphorylation and dephosphorylation of the H⁺-ATPase, are still unknown. In this study, we investigated in vitro phosphorylation and dephosphorylation of the H⁺-ATPase. The H⁺-ATPase was phosphorylated in the presence of ATP in the isolated microsome. An inhibitor of protein kinase, K-252a, had no effect on this phosphorylation. Dephosphrylation of the H⁺-ATPase was also found in the isolated microsome, and was inhibited by EDTA, but not by calyculin A, suggesting that type 2C protein phosphatase may catalyze dephosphorylation of the H⁺-ATPase. We will report analyses of the H⁺-ATPase complex by immunoprecipitation and gel filtration.