Molecular structure of the vacuolar hydrogen ion-translocating ATPase from a higher plant

An electrogenic H{dollar}sp+{dollar}-translocating ATPase in the vacuolar membrane (V-ATPase) of higher plants plays a central role in the acidification of vacuoles and transport of various solutes across vacuolar membranes. Among three major subunits common to all V-ATPases, the 70- and 60-kDa are catalytic and regulatory subunits respectively. Six copies of the N,N{dollar}spprime{dollar}-dicylohexylcarbodiimide-binding 16-kDa subunit are thought to form the H{dollar}sp+{dollar} pore. To elucidate the molecular structure of the V-ATPase, I have examined the organization of the V-ATPase subunits in the vacuolar membranes and determined the primary structure of the 16-kDa proteolipid subunit of the V-ATPase from oat (Avena sativa L. var Lang).; KI, a chaotropic reagent, and low ionic strength solutions solubilized the 72-, 60-, and 41-kDa subunits from vacuolar membranes but not the 16- and 13-kDa subunits. The subunits were identified by immunoblot with antibodies to the V-ATPase. These experiments provided direct evidence that the 72-, 60-, and 41-kDa polypeptides were peripheral subunits and the 16- and 13-kDa polypeptides were integral subunits of the V-ATPase. The subunit organization of the V-ATPase is similar to that of the F{dollar}sb1{dollar}F{dollar}sb{lcub}rm o{rcub}{dollar}-ATPase.; One cDNA (VATP-P1) encoding the proteolipid subunit with predicted 165 amino acids and molecular mass of 16,641 was obtained by screening oat root cDNA library with a synthetic oligonucleotide corresponding to a region of bovine proteolipid cDNA. Four membrane-spanning domains were predicted by hydropathy analysis and highly conserved among other eukaryotes such as bovine and yeast proteolipids. The partial sequences of three other cDNA clones (12, 54, and 93) encoded the same proteolipid but diverged in their codon usages and 3{dollar}spprime{dollar}-untranslated regions, indicating that they were derived from a small multigene family. A multigene family of the 16-kDa proteolipid in oat genome suggests that there are isoforms of the V-ATPase perhaps distributed in different tissues, cell types, or subcellular membranes.