title: Topology, glycosylation and conformational changes in the membrane domain of the vacuolar H+-ATPase a subunit.
abstract: Published topological models of the integral membrane a subunit of the vacuolar proton-translocating ATPase complex have not been in agreement with respect to either the number of transmembrane helices within the integral membrane domain, or their limits and orientations within the lipid bilayer. In the present work we have constructed a predictive model of the membrane insertion of the yeast a subunit, Vph1p, from a consensus of seven topology prediction algorithms. The model was tested experimentally using epitope tagging, green fluorescent protein fusion, and protease accessibility analysis in purified yeast vacuoles. Results suggest that a consensus prediction of eight transmembrane helices with both the amino-terminus and carboxyl-terminus in the cytoplasm is correct. Characterization of two glycosylation sites within the homologous mouse a subunit membrane domain further corroborates this topology. Moreover, the model takes into account published data on cytoplasmic and luminal accessibility of specific amino acids. Changes in the degree of protease accessibility in response to the V-ATPase substrate, MgATP, and the V-ATPase-specific inhibitor, concanamycin A, suggest that functional conformational changes occur in the large cytoplasmic loop between TM6 and TM7 of Vph1p. These data substantially confirm one topological model of the V-ATPase a subunit and support the notion that conformational changes occur within the membrane domain, possibly involving previously proposed axial rotation and/or linear displacement of TM7 in the proton transport cycle.
authors.affiliation: Faculty of Dentistry, Dental Research Institute, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Norbert
authors.initials: N
authors.lastname: Kartner
authors.forename: Yeqi
authors.initials: Y
authors.lastname: Yao
authors.forename: Ajay
authors.initials: A
authors.lastname: Bhargava
authors.forename: Morris F
authors.initials: MF
authors.lastname: Manolson
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004734:Energy Metabolism; D006031:Glycosylation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D025262:Vacuolar Proton-Translocating ATPases