Abstract
H+-pyrophosphatase (H+-PPase) generates a H+ gradient across endomembranes coupled with hydrolysis of pyrophosphate. In higher plants, a large amount of H+-PPase exists in vacuolar membranes and maintains the vacuolar acidic pH together with V-ATPase. However, it has been reported that H+-PPase is also localized in plasma membrane.
In this study, we inserted sGFP in a cytosolic loop of Arabidopsis type-I H+-PPase AtVHP1/AVP1 and transformed the construct with its own promoter into plant to determine intracellular localization. VHP1-sGFP expressed in plants had full enzyme activity and was fractionated into same fraction as endogenous VHP1 after sucrose density gradient centrifugation, indicating the adequate localization of VHP1-sGFP with function. VHP1-sGFP was detected in vacuolar membranes in all cells observed by CLSM. Strong fluorescence was observed in membranes of spherical structures within vacuoles, called bulbs. VHP1-sGFP was strongly expressed in almost all tissues, especially in growing tissues and vesicular bundles. In addition, there was a tendency that the expression level of VHP1-sGFP affect the number of bulbs.
