Vacuolar-type H⁺-ATPases are associated with the endoplasmic reticulum and provacuoles of root tip cells

To understand the origin of vacuolar H⁺-ATPases (V-ATPases) and their cellular functions, the subcellular location of V-H⁺-ATPases was examined immunologically in root cells of oat seedlings. A V-ATPase complex from oat roots consists of a large peripheral sector (V₁) that includes the 70-kD (A) catalytic and the 60-kD (B) regulatory subunits. The soluble V₁ complex, thought to be synthesized in the cytoplasm, is assembled with the membrane integral sector (V_O) at a yet undefined location. In mature cells, V-ATPase subunits A and B, detected in immunoblots with monoclonal antibodies (Mab) (7A5 and 2E7), were associated mainly with vacuolar membranes (20-22% sucrose) fractionated with an isopycnic sucrose gradient. However, in immature root tip cells, which lack large vacuoles, most of the V-ATPase was localized with the endoplasmic reticulum (ER) at 28 to 31% sucrose where a major ER-resident binding protein equilibrated. The peripheral subunits were also associated with membranes at 22% sucrose, at 31 to 34% sucrose (Golgi), and in plasma membranes at 38% sucrose. Immunogold labeling of root tip cells with Mab 2E7 against subunit B showed gold particles decorating the ER as well as numerous small vesicles (0.1-0.3 μm diameter), presumably provacuoles. The immunological detection of the peripheral subunit B on the ER supports a model in which the V₁ sector is assembled with the V_o on the ER. These results support the model in which the central vacuolar membrane originates ultimately from the ER. The presence of V-ATPases on several endomembranes indicates that this pump could participate in diverse functional roles.