V-type ATPase and pyrophosphatase in endomembranes of maize roots

Endomembrane fractions have been prepared from maize (Zea mays L.) roots. Electron microscopy and marker enzyme analysis indicate that they were highly purified. The endoplasmic reticulum (ER), the Golgi apparatus (GApp) and clathrln coated vesicles (CCV) were examined Immunologically for the presence of V-type H+ and pyrophosphatase polypeptides. In addition, these organelles were tested for the respective two enzyme activities as well as their capacity to pump protons in vitro. Our results demonstrate that, although neither enzyme activity nor proton pumping were measurable, the polypeptides of the two electrogenic pumps are clealy present in all three membrane types. Both extrinsic (70, 57, 44 kDa) and intrinsic (36, 16 kDa) V-ATPase subunits, as well as the Intrinsic 66 kDa subunit of the pyrophosphatase, were demonstrated, suggesting that the basic structure of these electrogenic pumps is already assembled at the level of the ER.