title: Vma8p-GFP fusions can be functionally incorporated into V-ATPase, suggesting structural flexibility at the top of V1.
abstract: The vacuolar ATPase (V-ATPase) complex of yeast (Saccharomyces cerevisiae) is comprised of two sectors, V(1) (catalytic) and V(O) (proton transfer). The hexameric (A(3)B(3)) cylinder of V(1) has a central cavity that must accommodate at least part of the rotary stalk of V-ATPase, a key component of which is subunit D (Vma8p). Recent electron microscopy (EM) data for the prokaryote V-ATPase complex (Thermus thermophilus) suggest that subunit D penetrates deeply into the central cavity. The functional counterpart of subunit D in mitochondrial F(1)F(O)-ATP synthase, subunit γ, occupies almost the entire length of the central cavity. To test whether the structure of yeast Vma8p mirrors that of subunit γ, we probed the location of the C-terminus of Vma8p by attachment of a large protein adduct, green fluorescent protein (GFP). We found that truncated Vma8p proteins lacking up to 40 C-terminal residues fused to GFP can be incorporated into functional V-ATPase complexes, and are able to support cell growth under alkaline conditions. We conclude that large protein adducts can be accommodated at the top of the central cavity of V(1) without compromising V-ATPase function, arguing for structural flexibility of the V(1) sector.
authors.affiliation: Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton campus, Victoria 3800, Australia; E-Mails: szczepan.nowakowski@eyeandear.org.au (S.N.); Dalibor.Mijaljica@monash.edu (D.M.); Mark.Prescott@monash.edu (M.P.).
authors.forename: Szczepan
authors.initials: S
authors.lastname: Nowakowski
authors.forename: Dalibor
authors.initials: D
authors.lastname: Mijaljica
authors.forename: Mark
authors.initials: M
authors.lastname: Prescott
authors.forename: Rodney J
authors.initials: RJ
authors.lastname: Devenish
mesh_terms: D055162:Biocatalysis; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016963:Thermus thermophilus / Q000201:enzymology; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D025262:Vacuolar Proton-Translocating ATPases