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.

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

“…In the earlier studies, [5][6][7][8] it was difficult to determine the orientation of the N-terminus of a-subunit isoforms, but it is now generally accepted that this domain is oriented toward the cytoplasm. Moreover, according to several recent electron microscopy studies of V-ATPases from different organisms 16,[38][39][40] and small-angle X-ray scattering data of NtpI 1-341 , 26 it was determined that the N-terminal domain of the a-subunit forms an elongated 1-shaped structure.…”

“…1 From a structural perspective, the topology of a-subunit is not understood. 4 For the yeast ''a'' subunit homolog Vph1, several topology models have been proposed over the years, [5][6][7][8] with the most recent model depicting eight transmembrane a-helices with both the Nterminal and C-terminal ends located on the cytoplasmic side of the membrane. 8 Although the location of the N-terminal region of the protein on the cytoplasmic site has been experimentally confirmed, its exact length has not been finally determined.…”

The vacuolar H+-ATPase: a universal proton pump of eukaryotes

“…Several of the putative transmembrane segments certainly contain conserved charged residues that could be involved in proton translocation, by analogy with the conserved acidic residue of the 16 kDa proteolipid. Recent mutagenesis work in yeast [151] has identified several key charged residues, mutation of which can affect both assembly and proton translocation function. In particular, mutation of conserved glutamates in the first and seventh putative segments, and of conserved histidine and arginine residues in segment 6, have profound effects, indicating a role for the Vph1 protein in the mechanism of proton translocation.…”

“…The Vph1p subunit of V o is reported to contain charged residues that are essential for proton translocation [151], leading to the supposition that Vph1p plays a role similar to that of subunit a in F o . Residues from both the 16 kDa proteolipid subunit c and Vph1p would contribute to a pathway of proton translocation.…”

Subunit Interactions in the Clathrin-coated Vesicle Vacuolar (H+)-ATPase Complex

“…After electrophoresis, the samples were electrophoretically transferred to nitrocellulose membranes for 16 h at 100 mA. The blots were then cut into strips, which were used in Western blot analysis as described previously (30). Protein A-Sepharose-purified antibodies were used at a dilution of 1:1000, and blots were developed using a chemiluminescent detection method from Kirkegaard and Perry Laboratories.…”