title: Structural analysis of the N-terminal domain of subunit a of the yeast vacuolar ATPase (V-ATPase) using accessibility of single cysteine substitutions to chemical modification.
abstract: The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V0) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V1V0 complex and the free V0 domain to identify residues protected from modification by the presence of V1. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V1 domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.
authors.affiliation: Department of Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Liberman
authors.forename: Kristina
authors.initials: K
authors.lastname: Cotter
authors.forename: James D
authors.initials: JD
authors.lastname: Baleja
authors.forename: Michael
authors.initials: M
authors.lastname: Forgac
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics
chemical_list: D025262:Vacuolar Proton-Translocating ATPases; D003545:Cysteine
keywords: ATPases; Bioenergetics; Biotin Maleimide; Chemical Modification; Cysteine Accessibility; Homology Modeling; Proton Transport; Subunit Interactions; Subunit a Structure; Vacuolar ATPase