title: Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector.
abstract: The membrane sector (Vo) of the proton pumping vacuolar ATPase (V-ATPase, V1Vo-ATPase) from Saccharomyces cerevisiae was purified to homogeneity, and its structure was characterized by EM of single molecules and two-dimensional crystals. Projection images of negatively stained Vo two-dimensional crystals showed a ring-like structure with a large asymmetric mass at the periphery of the ring. A cryo-EM reconstruction of Vo from single-particle images showed subunits a and d in close contact on the cytoplasmic side of the proton channel. A comparison of three-dimensional reconstructions of free Vo and Vo as part of holo V1Vo revealed that the cytoplasmic N-terminal domain of subunit a (aNT) must undergo a large conformational change upon enzyme disassembly or (re)assembly from Vo, V1, and subunit C. Isothermal titration calorimetry using recombinant subunit d and aNT revealed that the two proteins bind each other with a Kd of ~5 μm. Treatment of the purified Vo sector with 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] resulted in selective release of subunit d, allowing purification of a VoΔd complex. Passive proton translocation assays revealed that both Vo and VoΔd are impermeable to protons. We speculate that the structural change in subunit a upon release of V1 from Vo during reversible enzyme dissociation plays a role in blocking passive proton translocation across free Vo and that the interaction between aNT and d seen in free Vo functions to stabilize the Vo sector for efficient reassembly of V1Vo.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210.
authors.forename: Sergio
authors.initials: S
authors.lastname: Couoh-Cardel
authors.affiliation: From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210.
authors.forename: Elena
authors.initials: E
authors.lastname: Milgrom
authors.affiliation: From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210 wilkenss@upstate.edu.
authors.forename: Stephan
authors.initials: S
authors.lastname: Wilkens
mesh_terms: D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D020285:Cryoelectron Microscopy; D003461:Crystallography; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C556537:VMA2 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases
keywords: bioenergetics; cryo-EM; membrane transport; protein structure; proton pump; vacuolar ATPase