Abstract
H+-translocating pyrophosphatase (H+-PPases) is a unique enzyme implicated in the cellular pH homeostasis that develops an electrochemical H+ gradient across the membrane through pyrophosphate (PPi) hydrolysis. Phosphorylation is one of the posttranslational modifications (PTMs) of proteins and is considered a regulatory mechanism. In this study, the possible phosphorylation of the Vigna radiata H+-PPases (VrH+-PPases) isolated from the yeast heterologous expression system was revealed for the first time by using the Pro-Q Diamond staining. Furthermore, a phosphorylation site was identified at Ser45 of VrH+-PPases through mass spectroscopy. In addition, the application of casein kinase 2 (CK2) inhibitors resulted in the inhibition of phosphorylation of VrH+-PPases in yeast. Phosphorylation at Ser45 also enhanced the ion effects on VrH+-PPase. On the contrary, the type 2C protein phosphatase (PP2C) was able to dephosphorylate the VrH+-PPase obtained from heterologously expressing yeast. Our study provides new evidence for possible role of CK2 and PP2C in the kinase/phosphatase regulation of VrH+-PPases.