SITE-DIRECTED MUTAGENESIS OF THE 100-KDA SUBUNIT (VPH1P) OF THE YEASTVACUOLAR (H-ATPASE())

Vacuolar (H+) ATPases (V-ATPases) are multisubunit complexes responsib le for acidification of intracellular compartments in eukaryotic cells . V-ATPases possess a subunit of approximate molecular mass 100 kDa of unknown function that is composed of an amino-terminal hydrophilic do main and a carboxyl-terminal hydrophobic domain. To test whether the 1 00-kDa subunit plays a role in proton transport, site-directed mutagen esis of the VPH1 gene, which is one of two genes that encodes this sub unit in yeast, has been carried out in a strain lacking both endogenou s genes. Ten charged and twelve polar residues located in the seven pu tative transmembrane helices in the COOH-terminal domain of the molecu le were individually changed, and the effects on pro ton transport, AT Pase activity, and assembly of the yeast V-ATPase were measured. Two m utations (R735L and Q634L) in transmembrane helix 6 and at the border of transmembrane helix 5, respectively, showed greatly reduced levels of the 100 kDa subunit in the vacuolar membrane, suggesting that these mutations affected stability of the 100-kDa subunit. Two mutations, D 425N and K538A, in transmembrane helix 1 and at the border of transmem brane helix 3, respectively, showed reduced assembly of the V-ATPase, with the D425N mutation also reducing the activity of V-ATPase complex es that did assemble. Two mutations, H743A and K593A, in transmembrane helix 6 and at the border of transmembrane helix 4, respectively, hav e significantly greater effects on activity than on assembly, with pro ton transport and ATPase activity inhibited 40-60%. One mutation, E789 Q, in transmembrane helix 7, virtually completely abolished proton tra nsport and ATPase activity while having no effect on assembly. These r esults suggest that the 100-kDa subunit may be required for activity a s well as assembly of the V-ATPase complex and that several charged re sidues in the last four putative transmembrane helices of this subunit may play a role in proton transport.