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Biochem J. Sep 15, 2002; 366(Pt 3): 911–919.
PMCID: PMC1222818

Evidence that there are two copies of subunit c" in V0 complexes in the vacuolar H+-ATPase.

Abstract

The proton-translocating core of eukaryotic vacuolar H(+)-ATPase (V-ATPase), V(0) consists of a hexameric arrangement of transmembrane alpha-helices formed from the related polypeptides, subunit c and subunit c". The former is comprised of four transmembrane alpha-helices, whilst the latter has an extra transmembrane domain at its N-terminus. In addition, the fungal form of V(0) contains a minor subunit c-related polypeptide, subunit c'. All three are required for activity of the proton pump in Saccharomyces cerevisiae. We have introduced cysteine residues in the N-terminal extension of subunit c" in a cysteine-free form. All mutant forms are active in the V-ATPase from S. cerevisiae. Oxidation of vacuolar membranes containing the cysteine-replaced forms gave a cross-linked product of 42000Da. Analysis of this species showed it to be a dimeric form of subunit c", and further studies confirmed there are two copies of subunit c" in the V-ATPases in which it is present. Co-expression of double cysteine-replaced forms of both subunit c and c" gave rise to only homotypic cross-linked forms. Also, subunit c oligomeric complexes are present in vacuolar membranes in the absence of subunit c", consistent with previous observations showing hexameric arrangements of subunit c in gap-junction-like membranes. In vitro studies showed subunit c" can bind to subunit c and itself. The extent of binding can be increased by removal of the N-terminal domain of subunit c". This domain may therefore function to limit the copy number of subunit c" in V(0). A deletion study shows that the domain is essential for the activity of subunit c". The results can be combined into a model of V(0) which contains two subunit c" protomers with the extra transmembrane domain located toward the central pore. Thus the predicted stoichiometry of V(0) in which subunit c" is present is subunit c(3):subunit c'(1):subunit c"(2). On the basis of the mutational and binding studies, it seems likely that two copies of subunit c" are next to each other.

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Selected References

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  • Finbow ME, Harrison MA. The vacuolar H+-ATPase: a universal proton pump of eukaryotes. Biochem J. 1997 Jun 15;324(Pt 3):697–712. [PMC free article] [PubMed]
  • Stevens TH, Forgac M. Structure, function and regulation of the vacuolar (H+)-ATPase. Annu Rev Cell Dev Biol. 1997;13:779–808. [PubMed]
  • Nelson N, Harvey WR. Vacuolar and plasma membrane proton-adenosinetriphosphatases. Physiol Rev. 1999 Apr;79(2):361–385. [PubMed]
  • Vänänen HK, Karhukorpi EK, Sundquist K, Wallmark B, Roininen I, Hentunen T, Tuukkanen J, Lakkakorpi P. Evidence for the presence of a proton pump of the vacuolar H(+)-ATPase type in the ruffled borders of osteoclasts. J Cell Biol. 1990 Sep;111(3):1305–1311. [PMC free article] [PubMed]
  • Swallow CJ, Grinstein S, Sudsbury RA, Rotstein OD. Relative roles of Na+/H+ exchange and vacuolar-type H+ ATPases in regulating cytoplasmic pH and function in murine peritoneal macrophages. J Cell Physiol. 1993 Dec;157(3):453–460. [PubMed]
  • Boyer PD. The ATP synthase--a splendid molecular machine. Annu Rev Biochem. 1997;66:717–749. [PubMed]
  • Noji H, Yasuda R, Yoshida M, Kinosita K., Jr Direct observation of the rotation of F1-ATPase. Nature. 1997 Mar 20;386(6622):299–302. [PubMed]
  • Jones PC, Jiang W, Fillingame RH. Arrangement of the multicopy H+-translocating subunit c in the membrane sector of the Escherichia coli F1F0 ATP synthase. J Biol Chem. 1998 Jul 3;273(27):17178–17185. [PubMed]
  • Stock D, Leslie AG, Walker JE. Molecular architecture of the rotary motor in ATP synthase. Science. 1999 Nov 26;286(5445):1700–1705. [PubMed]
  • Seelert H, Poetsch A, Dencher NA, Engel A, Stahlberg H, Müller DJ. Structural biology. Proton-powered turbine of a plant motor. Nature. 2000 May 25;405(6785):418–419. [PubMed]
  • Girvin ME, Rastogi VK, Abildgaard F, Markley JL, Fillingame RH. Solution structure of the transmembrane H+-transporting subunit c of the F1F0 ATP synthase. Biochemistry. 1998 Jun 23;37(25):8817–8824. [PubMed]
  • Harrison MA, Finbow ME, Findlay JB. Postulate for the molecular mechanism of the vacuolar H(+)-ATPase (hypothesis). Mol Membr Biol. 1997 Jan-Mar;14(1):1–3. [PubMed]
  • Hirata R, Graham LA, Takatsuki A, Stevens TH, Anraku Y. VMA11 and VMA16 encode second and third proteolipid subunits of the Saccharomyces cerevisiae vacuolar membrane H+-ATPase. J Biol Chem. 1997 Feb 21;272(8):4795–4803. [PubMed]
  • Powell B, Graham LA, Stevens TH. Molecular characterization of the yeast vacuolar H+-ATPase proton pore. J Biol Chem. 2000 Aug 4;275(31):23654–23660. [PubMed]
  • Peters C, Bayer MJ, Bühler S, Andersen JS, Mann M, Mayer A. Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature. 2001 Feb 1;409(6820):581–588. [PubMed]
  • Israël M, Lesbats B, Morel N, Manaranche R, Gulik-Krzywicki T, Dedieu JC. Reconstitution of a functional synaptosomal membrane possessing the protein constituents involved in acetylcholine translocation. Proc Natl Acad Sci U S A. 1984 Jan;81(1):277–281. [PMC free article] [PubMed]
  • Finbow ME, Shuttleworth J, Hamilton AE, Pitts JD. Analysis of vertebrate gap junction protein. EMBO J. 1983;2(9):1479–1486. [PMC free article] [PubMed]
  • Finbow ME, Buultjens TE, Lane NJ, Shuttleworth J, Pitts JD. Isolation and characterisation of arthropod gap junctions. EMBO J. 1984 Oct;3(10):2271–2278. [PMC free article] [PubMed]
  • Finbow ME, Eliopoulos EE, Jackson PJ, Keen JN, Meagher L, Thompson P, Jones P, Findlay JB. Structure of a 16 kDa integral membrane protein that has identity to the putative proton channel of the vacuolar H(+)-ATPase. Protein Eng. 1992 Jan;5(1):7–15. [PubMed]
  • Holzenburg A, Jones PC, Franklin T, Pali T, Heimburg T, Marsh D, Findlay JB, Finbow ME. Evidence for a common structure for a class of membrane channels. Eur J Biochem. 1993 Apr 1;213(1):21–30. [PubMed]
  • Finbow ME, Pitts JD, Goldstein DJ, Schlegel R, Findlay JB. The E5 oncoprotein target: a 16-kDa channel-forming protein with diverse functions. Mol Carcinog. 1991;4(6):441–444. [PubMed]
  • John SA, Saner D, Pitts JD, Holzenburg A, Finbow ME, Lal R. Atomic force microscopy of arthropod gap junctions. J Struct Biol. 1997 Oct;120(1):22–31. [PubMed]
  • Harrison MA, Murray J, Powell B, Kim YI, Finbow ME, Findlay JB. Helical interactions and membrane disposition of the 16-kDa proteolipid subunit of the vacuolar H(+)-ATPase analyzed by cysteine replacement mutagenesis. J Biol Chem. 1999 Sep 3;274(36):25461–25470. [PubMed]
  • Harrison M, Powell B, Finbow ME, Findlay JB. Identification of lipid-accessible sites on the nephrops 16-kDa proteolipid incorporated into a hybrid vacuolar H(+)-ATPase: site-directed labeling with N-(1-Pyrenyl)cyclohexylcarbodiimide and fluorescence quenching analysis. Biochemistry. 2000 Jun 27;39(25):7531–7537. [PubMed]
  • Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. [PubMed]
  • Dunlop J, Jones PC, Finbow ME. Membrane insertion and assembly of ductin: a polytopic channel with dual orientations. EMBO J. 1995 Aug 1;14(15):3609–3616. [PMC free article] [PubMed]
  • Sun-Wada GH, Murakami H, Nakai H, Wada Y, Futai M. Mouse Atp6f, the gene encoding the 23-kDa proteolipid of vacuolar proton translocating ATPase. Gene. 2001 Aug 22;274(1-2):93–99. [PubMed]
  • Leitch B, Finbow ME. The gap junction-like form of a vacuolar proton channel component appears not to be an artifact of isolation: an immunocytochemical localization study. Exp Cell Res. 1990 Oct;190(2):218–226. [PubMed]
  • Uchida E, Ohsumi Y, Anraku Y. Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem. 1985 Jan 25;260(2):1090–1095. [PubMed]
  • Ashby AD, Meagher L, Campo MS, Finbow ME. E5 transforming proteins of papillomaviruses do not disturb the activity of the vacuolar H(+)-ATPase. J Gen Virol. 2001 Oct;82(Pt 10):2353–2362. [PubMed]
  • Nishi T, Kawasaki-Nishi S, Forgac M. Expression and localization of the mouse homologue of the yeast V-ATPase 21-kDa Subunit c" (Vma16p). J Biol Chem. 2001 Sep 7;276(36):34122–34130. [PubMed]

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