title: A site-directed cross-linking approach to the characterization of subunit E-subunit G contacts in the vacuolar H+-ATPase stator.
abstract: To operate as a rotary motor, the ATP-hydrolyzing domain of the vacuolar H(+)-ATPase must be connected to a fixed structure in its membrane-bound proton pump domain by a mechanical stator. Although low-resolution structural data and spectroscopic analysis indicate that a filament-like subunit E/subunit G heterodimer performs this role, more detailed information about the relative arrangement of these subunits is limited. We have used a site-directed cross-linking approach to show that, in both bacterial and yeast V-type ATPases, the N-terminal alpha-helical segments of the G and E subunits are closely aligned over a distance of up to 40 A. Furthermore, cross-linking coupled to mass spectrometry shows that the C-terminal end of G is anchored at the C-terminal globular domain of subunit E. These data are consistent with a stator model comprising two approximately 150 A long parallel alpha-helices linked to each other at both ends, stabilized by a coiled-coil arrangement and capped by the globular C-terminal domain of E that connects the cytoplasmic end of the helical structure to the V-ATPase catalytic domain.
authors.affiliation: Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, UK.
authors.forename: Richard P O
authors.initials: RP
authors.lastname: Jones
authors.forename: Lyndsey J
authors.initials: LJ
authors.lastname: Durose
authors.forename: Clair
authors.initials: C
authors.lastname: Phillips
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Keen
authors.forename: John B C
authors.initials: JB
authors.lastname: Findlay
authors.forename: Michael A
authors.initials: MA
authors.lastname: Harrison
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D016983:Enterococcus / Q000201:enzymology / Q000235:genetics; D015151:Immunoblotting; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D025262:Vacuolar Proton-Translocating ATPases