Site-directed Mutagenesis of the 100-kDa Subunit (Vph1p) of the Yeast Vacuolar (H+)-ATPase

Abstract

Vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible 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 domain and a carboxyl-terminal hydrophobic domain. To test whether the 100-kDa subunit plays a role in proton transport, site-directed mutagenesis of the VPH1 gene, which is one of two genes that encodes this subunit in yeast, has been carried out in a strain lacking both endogenous genes. Ten charged and twelve polar residues located in the seven putative transmembrane helices in the COOH-terminal domain of the molecule were individually changed, and the effects on proton transport, ATPase activity, and assembly of the yeast V-ATPase were measured. Two mutations (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, D425N and K538A, in transmembrane helix 1 and at the border of transmembrane helix 3, respectively, showed reduced assembly of the V-ATPase, with the D425N mutation also reducing the activity of V-ATPase complexes that did assemble. Two mutations, H743A and K593A, in transmembrane helix 6 and at the border of transmembrane helix 4, respectively, have significantly greater effects on activity than on assembly, with proton transport and ATPase activity inhibited 40-60%. One mutation, E789Q, in transmembrane helix 7, virtually completely abolished proton transport and ATPase activity while having no effect on assembly. These results suggest that the 100-kDa subunit may be required for activity as well as assembly of the V-ATPase complex and that several charged residues in the last four putative transmembrane helices of this subunit may play a role in proton transport.