A 100 kDa polypeptide associates with the VO sector but not with the active oat vacuolar H+-ATPase suggesting a role in assembly

Vacuolar H+-ATPase (V-ATPase) is responsible for acidifying endomembranc compartments in eukaryotic cells. Although a 100-kDa subunit is common to many V-ATPases, it is absent from a purified and active pump of oat. Im munostaining revealed a Vphlp-related polypeptide in oat membranes, thus tho role of this polypeptide was investigated. Membrane proteins were detergent solubilized and size-fractionated, and V-ATPase subunits were identified by immunostaining. A 100-kDa polypeptide was not associated with the fully assembled ATPase; however it was part of a 250 kDa V0 complex including subunits of 36- and 16-kDa. coimmunoprecipitation with a monoclonal anti body against A subunit indicated that free Vi subcomplexes existed in the cytosol. The free Vi subcomplex became attached to the detergent-solubilized V0 sector after mixing, and an antibody against subunit A coprecipitated all Vj and Yû subunits except for the 100-kDa polypeptide. The absence of this polypeptide from the active enzyme demonstrates that, unlike the yeast Vphlp, the 100-kDa polypeptide in oat is not required for activity. Its association with the free V0 sector at the endoplasmic reticulum would support a role of this protein in the assembly of the VD subcomplex and of the entire V-ATPase.