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.

Molecular mechanisms of cutis laxa– and distal renal tubular acidosis–causing mutations in V-ATPase a subunits, ATP6V0A2 and ATP6V0A4

“…This model is based on a synthesis of data derived from cryo-EM 3D reconstruction, evolutionary covariance mapping of key residues, and low resolution X-ray crystallography. It confirms that the a subunit membrane domain consists of 8 TMs, as has been previously shown (2,12), with TM7 and TM8 highly tilted and forming an interface with the V0 rotor c-ring that enables proton translocation at the a subunit/c-ring interface.…”

“…We have shown ELII is the site of N-glycosylation within subunit a1-a4 (25,26) indicating that ELII is luminal/extracellular. In contrast, we and others have shown that the C-terminal domain is cytoplasmic (12,25,26).Comparing the accessibility of either epitope in permeabilized vs nonpermeabilized cells can determine if a4 is expressed on the cell surface. In permeabilized cells, one would expect that both cytoplasmic and extracellular epitopes would be assessable to fluorescently-labeled antibodies; in nonpermeabilized cells, only HA on the extracellular EL2, would be available.…”

“…A 3D representation of a4 G820R was generated after substituting Gly 820 with Arg, using the YASARA FoldX plug-in. (12). Red highlights indicate a.a. affected by human disease-causing mutations (noted above alignments).…”

“…V1 is responsible for ATP hydrolysis that provides the energy to rotate a central shaft that powers proton translocation (3). V0 contains a coupled rotor that carries protons for transport through a proton channel pathway formed largely by the c. 100-kDa a subunit (2,12). The a subunit is the largest V-ATPase subunit, and in mammals there are four isoforms, a1-a4.…”

The Endoplasmic Reticulum Is the Main Membrane Source for Biogenesis of the Lytic Vacuole inArabidopsis

“…Nevertheless, we have shown here that in the pat2-1 mutant, lacking the b-subunit of the AP3 complex (Feraru et al, 2010), VHA-a3 and AVP1/VHP1 still reach their destination pointing to the existence of a third route to the tonoplast of the LV that might completely bypass the Golgi apparatus. Although mammalian isoforms of subunit a have been shown to be modified by N-glycosylation (Kartner et al, 2013), VHA-a3 lacks predicted N-glycosylation sites and passage through the Golgi is thus not required.…”