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• W2083872341 abstract "The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that acidifies intracellular organelles in eukaryotes. Similar to the F-type ATP synthase (FATPase), the V-ATPase is composed of two subcomplexes, V1 and V0. Hydrolysis of ATP in the V1 subcomplex is tightly coupled to proton translocation accomplished by the V0 subcomplex, which is composed of five unique subunits (a, d, c, c′, and c″). Three of the subunits, subunit c (Vma3p), c′ (Vma11p), and c″ (Vma16p), are small highly hydrophobic integral membrane proteins called “proteolipids” that share sequence similarity to the F-ATPase subunit c. Whereas subunit c from the F-ATPase spans the membrane bilayer twice, the V-ATPase proteolipids have been modeled to have at least four transmembrane-spanning helices. Limited proteolysis experiments with epitope-tagged copies of the proteolipids have revealed that the N and the C termini of c (Vma3p) and c′ (Vma11p) were in the lumen of the vacuole. Limited proteolysis of epitope-tagged c″ (Vma16p) indicated that the N terminus is located on the cytoplasmic face of the vacuole, whereas the C terminus is located within the vacuole. Furthermore, a chimeric fusion between Vma16p and Vma3p, Vma16-Vma3p, was found to assemble into a fully functional V-ATPase complex, further supporting the conclusion that the C terminus of Vma16p resides within the lumen of the vacuole. These results indicate that subunits c and c′ have four transmembrane segments with their N and C termini in the lumen and that c″ has five transmembrane segments, with the N terminus exposed to the cytosol and the C terminus lumenal. The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that acidifies intracellular organelles in eukaryotes. Similar to the F-type ATP synthase (FATPase), the V-ATPase is composed of two subcomplexes, V1 and V0. Hydrolysis of ATP in the V1 subcomplex is tightly coupled to proton translocation accomplished by the V0 subcomplex, which is composed of five unique subunits (a, d, c, c′, and c″). Three of the subunits, subunit c (Vma3p), c′ (Vma11p), and c″ (Vma16p), are small highly hydrophobic integral membrane proteins called “proteolipids” that share sequence similarity to the F-ATPase subunit c. Whereas subunit c from the F-ATPase spans the membrane bilayer twice, the V-ATPase proteolipids have been modeled to have at least four transmembrane-spanning helices. Limited proteolysis experiments with epitope-tagged copies of the proteolipids have revealed that the N and the C termini of c (Vma3p) and c′ (Vma11p) were in the lumen of the vacuole. Limited proteolysis of epitope-tagged c″ (Vma16p) indicated that the N terminus is located on the cytoplasmic face of the vacuole, whereas the C terminus is located within the vacuole. Furthermore, a chimeric fusion between Vma16p and Vma3p, Vma16-Vma3p, was found to assemble into a fully functional V-ATPase complex, further supporting the conclusion that the C terminus of Vma16p resides within the lumen of the vacuole. These results indicate that subunits c and c′ have four transmembrane segments with their N and C termini in the lumen and that c″ has five transmembrane segments, with the N terminus exposed to the cytosol and the C terminus lumenal. The vacuolar H+-ATPase (V-ATPase) 1The abbreviations used are: V-ATPase, vacuolar ATPase; YEPD, yeast extract/peptone/dextrose; HA, hemagglutinin; Mes, 4-morpholineethanesulfonic acid; TRITC, tetramethylrhodamine isothiocyanate; CPY, carboxypeptidase Y. is a multisubunit enzyme responsible for the lumenal acidification of cellular organelles (1Anraku Y. Hirata R. Wada Y. Ohya Y. J. Exp. Biol. 1992; 172: 67-81Crossref PubMed Google Scholar). Organelle acidification is essential for a variety of cellular processes such as receptor-mediated endocytosis, activation of proteases, and proton-coupled transport of small molecules and ions (2Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (523) Google Scholar, 3Graham L.A. Flannery A.R. Stevens T.H. J. Bioenerg. Biomembr. 2003; 35: 301-312Crossref PubMed Scopus (84) Google Scholar, 4Nishi T. Forgac M. Nat. Rev. Mol. Cell Biol. 2002; 3: 94-103Crossref PubMed Scopus (1005) Google Scholar). Similar to the F-type ATP synthase, the V-ATPase is composed of two distinct sectors; a peripheral catalytic complex (V1) and a membrane associated complex (V0). The peripheral catalytic V1 subcomplex is a 560-kDa domain composed of eight subunits (A, B, C, D, E, F, G, and H). Its catalytic core is composed of a hexameric ring of alternating A (69 kDa) and B (57 kDa) subunits (5Arai H. Terres G. Pink S. Forgac M. J. Biol. Chem. 1988; 263: 8796-8802Abstract Full Text PDF PubMed Google Scholar). Both the A and B subunits contain consensus sequences for ATP binding domains, where subunit A has been ascribed with the catalytic function (6Hirata R. Ohsumk Y. Nakano A. Kawasaki H. Suzuki K. Anraku Y. J. Biol. Chem. 1990; 265: 6726-6733Abstract Full Text PDF PubMed Google Scholar), and subunit B is proposed to have a regulatory role (7Vasilyeva E. Liu Q. MacLeod K.J. Baleja J.D. Forgac M. J. Biol. Chem. 2000; 275: 255-260Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The role of the other V1 subunits is not so clear. It is thought that the central stalk may be composed of D (32 kDa) and F (14 kDa) subunits (8Arata Y. Nishi T. Kawasaki-Nishi S. Shao E. Wilkens S. Forgac M. Biochim. Biophys. Acta. 2002; 1555: 71-74Crossref PubMed Scopus (40) Google Scholar, 9Arata Y. Baleja J.D. Forgac M. Biochemistry. 2002; 41: 11301-11307Crossref PubMed Scopus (77) Google Scholar). The stator region is thought to be composed of E (27 kDa), G (13 kDa), C (42 kDa), and H (54 kDa) subunits (9Arata Y. Baleja J.D. Forgac M. Biochemistry. 2002; 41: 11301-11307Crossref PubMed Scopus (77) Google Scholar, 10Keenan Curtis K. Kane P.M. J. Biol. Chem. 2002; 277: 2716-2724Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 11Charsky C.M. Schumann N.J. Kane P.M. J. Biol. Chem. 2000; 275: 37232-37239Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 12Arata Y. Baleja J.D. Forgac M. J. Biol. Chem. 2002; 277: 3357-3363Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 13Tomashek J.J. Graham L.A. Hutchins M.U. Stevens T.H. Klionsky D.J. J. Biol. Chem. 1997; 272: 26787-26793Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). All subunits are required for a fully assembled complex with the exception of the H subunit; however, the H subunit is required for a functional complex (14Ho M.N. Hirata R. Umemoto N. Ohya Y. Takatsuki A. Stevens T.H. Anraku Y. J. Biol. Chem. 1993; 268: 18286-18292Abstract Full Text PDF PubMed Google Scholar). The membrane-associated V0 subcomplex is composed of five subunits (a, c, c′, c″, and d). There is a ring of the proteolipid subunits Vma3p (16.5 kDa, subunit c), Vma11p (17 kDa, subunit c′), and Vma16p (23 kDa, subunit c″), and each proteolipid subunit contains a conserved acidic residue required for proton translocation (15Hirata R. Graham L.A. Takatsuki A. Stevens T.H. Anraku Y. J. Biol. Chem. 1997; 272: 4795-4803Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The proteolipid ring contains one copy each of the c′ and c″ subunits (16Powell B. Graham L.A. Stevens T.H. J. Biol. Chem. 2000; 275: 23654-23660Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), with 4-5 copies of subunit c (4Nishi T. Forgac M. Nat. Rev. Mol. Cell Biol. 2002; 3: 94-103Crossref PubMed Scopus (1005) Google Scholar, 5Arai H. Terres G. Pink S. Forgac M. J. Biol. Chem. 1988; 263: 8796-8802Abstract Full Text PDF PubMed Google Scholar). The a (100-kDa) subunit has two isoforms, Vph1p and Stv1p. The two isoforms specify to which organelles the V-ATPase complex is routed; the Vph1p containing V-ATPase complex localizes to the vacuole, whereas the Stv1p-containing complex remains in the Golgi/endosome membranes (17Kawasaki-Nishi S. Bowers K. Nishi T. Forgac M. Stevens T.H. J. Biol. Chem. 2001; 276: 47411-47420Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 18Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar). The a subunit contains two domains, a hydrophilic N terminus domain believed to form part of the stator and a hydrophobic carboxyl domain, which also forms part of the proton pore (19Leng X.H. Manolson M.F. Liu Q. Forgac M. J. Biol. Chem. 1996; 271: 22487-22493Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 20Leng X.H. Nishi T. Forgac M. J. Biol. Chem. 1999; 274: 14655-14661Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 21Jackson D.D. Stevens T.H. J. Biol. Chem. 1997; 272: 25928-25934Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The function of the d (36 kDa) subunit is currently unknown, but recently a x-ray crystal structure has been published, in which the authors propose that the d subunit may cap the top of the proteolipid ring (22Iwata M. Imamura H. Stambouli E. Ikeda C. Tamakoshi M. Nagata K. Makyio H. Hankamer B. Barber J. Yoshida M. Yokoyama K. Iwata S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 59-64Crossref PubMed Scopus (115) Google Scholar). Like the F-type ATP synthase, the V-ATPase has been shown to work by a rotary mechanism. ATP is hydrolyzed by the A subunit causing a conformation change and, thus, rotating the central stalk composed of F and D (23Imamura H. Nakano M. Noji H. Muneyuki E. Ohkuma S. Yoshida M. Yokoyama K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2312-2315Crossref PubMed Scopus (170) Google Scholar, 24Hirata T. Iwamoto-Kihara A. Sun-Wada G.H. Okajima T. Wada Y. Futai M. J. Biol. Chem. 2003; 278: 23714-23719Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The stalk is fixed to the ring of c subunits and causes it to rotate as well (25Yokoyama K. Nakano M. Imamura H. Yoshida M. Tamakoshi M. J. Biol. Chem. 2003; 278: 24255-24258Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The c subunit ring rotates relative to the stator, thus pumping protons with a rotary mechanism. Unlike the F-type ATP synthase, the VATPase functions physiologically to pump protons across the lipid bilayer and not to synthesize ATP. NMR and x-ray crystallography studies have shown that the F-type ATP synthase 8-kDa-subunit forms an α-helical hairpin with two transmembrane helices such that the N termini and the C termini oriented away from the F1 subcomplex (26Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1093) Google Scholar, 27Dmitriev O.Y. Jones P.C. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7785-7790Crossref PubMed Scopus (107) Google Scholar, 28Girvin M.E. Rastogi V.K. Abildgaard F. Markley J.L. Fillingame R.H. Biochemistry. 1998; 37: 8817-8824Crossref PubMed Scopus (272) Google Scholar, 29Jones P.C. Fillingame R.H. J. Biol. Chem. 1998; 273: 29701-29705Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). There is no high resolution structure of the c subunits from the V-ATPase. However, based upon the similarity to the F-type ATP synthase c subunit and hydropathy plots, Vma3p and Vma11p can be modeled to have four transmembrane helices and Vma16p to have five transmembrane helices (3Graham L.A. Flannery A.R. Stevens T.H. J. Bioenerg. Biomembr. 2003; 35: 301-312Crossref PubMed Scopus (84) Google Scholar). The VATPase subunits may have arisen from a gene duplication event of a common c subunit ancestor (30Mandel M. Moriyama Y. Hulmes J.D. Pan Y.C. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5521-5524Crossref PubMed Scopus (239) Google Scholar, 31Noumi T. Beltran C. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1938-1942Crossref PubMed Scopus (128) Google Scholar, 32Nelson H. Nelson N. FEBS Lett. 1989; 247: 147-153Crossref PubMed Scopus (124) Google Scholar). This would suggest that the orientation of the termini of Vma3p and Vma11p would lie within the lumen of the vacuole, whereas Vma16p would have either the N terminus or the C terminus in the lumen of the vacuole. Previous studies have suggested that Vma16p (c″) has either four (33Nishi T. Kawasaki-Nishi S. Forgac M. J. Biol. Chem. 2003; 278: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) or five (34Gibson L.C. Cadwallader G. Finbow M.E. Biochem. J. 2002; 366: 911-919Crossref PubMed Google Scholar) transmembrane regions, and these studies came to different conclusions about the requirement for the first putative transmembrane helix of Vma16p. In this paper we describe the molecular and biochemical approach that we used to address the topology of Vma3p, Vma11p, and Vma16p. We have attached epitope tags to the N and C termini of these three subunits and used protease protection assays to determine the orientation of the termini. Through immunoblot analysis of the limited proteolysis we have determined that the N and C termini of Vma3p and Vma11p are located within the lumen of the vacuole, whereas the N terminus of Vma16p is located in the cytoplasm, and the C terminus is in the lumen. In addition, we have also constructed chimeric fusions of Vma16p and Vma3p that further support our topological model of Vma16p. Strains and Culture Conditions—The yeast strains and plasmids used in this study are listed in Tables I and II. All yeast strains were cultured in SD minimal media (0.67% yeast nitrogen base, 2% dextrose) supplemented with the appropriate amino acids or YEPD medium buffered to pH 5.0 using 50 mm succinate/phosphate. To test for a Vma- phenotype, saturated cultures were diluted to an A600 = 0.25. Serial dilutions of the diluted culture were applied to pH 7.5 YEPD plus 100 mm CaCl2 media and allowed to grow overnight before observing results.Table ISaccharomyces cervevisiae strains used in this studyStrainGenotypeSourceSF838-1DαMATα, ade6, leu2-3, 112, ura3-52, pep4-3, his4-519, gal 2Ref. 51Rothman J.H. Stevens T.H. Cell. 1986; 47: 1041-1051Abstract Full Text PDF PubMed Scopus (299) Google ScholarLGY9MATα, ade6, leu2-3, 112, ura3-52, pep4-3, his4-519, gal 2, uma16::LEU2Ref. 52Bowman E.J. Graham L.A. Stevens T.H. Bowman B.J. J. Biol. Chem. 2004; 279: 33131-33138Abstract Full Text Full Text PDF PubMed Scopus (115) Google ScholarRHA107MATα, ade6, leu2-3, 112, ura3-52, pep4-3, his4-519, gal 2, uma11::LEU2Gift from R. HirataLGY101MATα, ade6, leu2-3, 112, ura3-52, pep4-3, his4-519, gal 2, uma3::LEU2Ref. 52Bowman E.J. Graham L.A. Stevens T.H. Bowman B.J. J. Biol. Chem. 2004; 279: 33131-33138Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar Open table in a new tab Table IIS. plasmids used in this studyPlasmidsDescriptionSourcepRS316Centromere based, low copy plasmid (pCEN-URA)Ref. 53Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarpLG931.8-kbakb, kilobases.PstI-EcoRI VMA3::1×HA subcloned into pRS316Ref. 52Bowman E.J. Graham L.A. Stevens T.H. Bowman B.J. J. Biol. Chem. 2004; 279: 33131-33138Abstract Full Text Full Text PDF PubMed Scopus (115) Google ScholarpLG871.8-kb EcoRV-SpeI VMA11::3×HA subcloned into pRS316Ref. 52Bowman E.J. Graham L.A. Stevens T.H. Bowman B.J. J. Biol. Chem. 2004; 279: 33131-33138Abstract Full Text Full Text PDF PubMed Scopus (115) Google ScholarpLG841.9-kb BalI-EcoRI VMA16::3×HA subcloned into pRS316Ref. 52Bowman E.J. Graham L.A. Stevens T.H. Bowman B.J. J. Biol. Chem. 2004; 279: 33131-33138Abstract Full Text Full Text PDF PubMed Scopus (115) Google ScholarpAJ821.9-kb BalI-EcoRI c-myc::VMA16::3×HA subcloned into pRS316This studypLG961.9-kb BalI-EcoRI c-myc::vma16Δ12-38::3×HA subcloned into pRS316This studypAF2711.8-kb PstI-EcoRI 1×HA::VMA3 subcloned into pRS316This studypLG1711.8-kb EcoRV-SpeI 1×HA::VMA11 subcloned into pRS316This studypLG146VMA16::3×HA::VMA3 dimer under control of VMA3 promoter subcloned into pRS316This studypLG145VMA3::3×HA::VMA16 dimer under control of VMA3 promoter subcloned into pRS316This studya kb, kilobases. Open table in a new tab Plasmid Construction and Epitope Tagging—DNA sequence encoding a single c-Myc epitope was introduced immediately after the start codon of pLG84 to generate pAJ82 by PCR using outward facing oligonucleotides. PCR using outward-facing oligonucleotides was also used to delete base pairs 34-114 from pAJ82 to generate pLG96. QuikChange mutagenesis was used to introduce an MluI site (A/CGCGT) immediately after the start codon of VMA3 in plasmid pRHA316 (16Powell B. Graham L.A. Stevens T.H. J. Biol. Chem. 2000; 275: 23654-23660Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). PCR was used to construct a 1×HA::VMA3 from pLG93 that was flanked by an MluI 5′ site and a BglII 3′ site. The MluI/BglII-digested PCR fragment was then ligated into a MluI/BglII-digested pLG93 to create pAF271. QuikChange mutagenesis (Stratagene) was used to introduce a BglII site ((A/G)ATCT) immediately after the start codon in pRS316VMA11(pNUVA388) to generate pLG159. Complementary oligonucleotides were synthesized so that when annealed together they formed an oligonucleotide duplex encoding a single HA epitope with BglII ends. The oligonucleotide duplex was ligated into BglII-digested pLG159 to generate pLG171. QuikChange mutagenesis was used to introduce an MluI site (A/CGCGT) immediately after the start codon in pLG93 to generate pLG101. A VMA16::3×HA fragment was generated from pLG84 by PCR using oligonucleotides that introduced MluI sites on the ends of the PCR fragment. MluI-digested pLG101 was ligated with MluI-digested VMA16::3×HA PCR fragment to generate pLG146. A BglII site was inserted immediately after the start codon in pRS316VMA16::c-myc (pLG33) to generate pLG94. A 3×HA BglII fragment from BJ7122 was ligated into digested pLG33 to form pLG94. A 3×HA::VMA16 PCR fragment was generated from pLG94 by PCR using oligonucleotides that introduce MluI sites on the ends of the fragment. QuikChange mutagenesis was used to also introduce a MluI site immediately before the stop codon of pRS316VMA3 (pRHA313) to generate pLG100. The 3×HA::VMA16 MluI-digested PCR fragment was ligated with digested pLG100 to create pLG145. Proteolytic Protection Assays—Intact vacuolar vesicles were isolated using a previously described procedure with modifications (21Jackson D.D. Stevens T.H. J. Biol. Chem. 1997; 272: 25928-25934Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 35Uchida E. Ohsumi Y. Anraku Y. J. Biol. Chem. 1985; 260: 1090-1095Abstract Full Text PDF PubMed Google Scholar). Briefly, yeast were grown to log phase of A600 = 4.0 in YEPD medium buffered to pH 5.0, cells were converted to spheroplast, and lysed with a loosely fitting Dounce homogenizer in Buffer A (10 mm Mes/Tris, pH 6.9, 0.1 mm MgCl2, 12% Ficoll). The lysate was centrifuged at 60,000 × g for 45 min in a swinging bucket rotor. The floating white layer containing the vacuoles were removed with a weigh spatula and resuspended in 10 ml of modified buffer 88 (M88) (20 mm HEPES, pH 6.8, 150 mm potassium acetate, 1 mm magnesium acetate, 1 mm CaCl2, 250 mm sorbitol). The vacuoles were recovered by centrifugation at 13,000 × g, washed twice in M88 buffer, and brought up to a final protein concentration of 4 mg/ml. Samples containing 1 mg of protein were treated at 4 °C with trypsin (0.5 mg/ml) and proteinase K (0.5 mg/ml) in the absence or presence of 1% Triton X-100 for 10 min. The reactions were quenched by the addition of an equal volume of 40% trichloroacetic acid. The samples were pelleted at 13,000 × g and washed with an equal volume of ice-cold acetone. The pellets were resuspended in Thorner buffer (8 m urea, 5% SDS, 40 mm Tris, pH 6.8, 0.1 mm EDTA, 0.4 mg/ml bromphenol blue, β-mercaptoethanol added fresh to 5%), and 10 μg of protein was loaded per lane of a SDS-PAGE. Protein Preparation, SDS-PAGE, and Immunoblot Analysis—Whole cell extracts and vacuolar membranes were prepared as described previously (35Uchida E. Ohsumi Y. Anraku Y. J. Biol. Chem. 1985; 260: 1090-1095Abstract Full Text PDF PubMed Google Scholar, 36Conibear E. Stevens T.H. Methods Enzymol. 2002; 351: 408-432Crossref PubMed Scopus (45) Google Scholar). SDS-PAGE analysis was performed. Proteins were transferred to 0.2-μm nitrocellulose and probed with either an affinity-purified monoclonal anti-HA (Covance Research Products Inc.) used at 1:3000, the anti-Myc 9E10 described previously (37Conibear E. Stevens T.H. Mol. Biol. Cell. 2000; 11: 305-323Crossref PubMed Scopus (234) Google Scholar) used at 1:5, antibodies 10D7 or 7B1 used to probe for Vph1p at 1:500 (Molecular Probes), the monoclonal antibody 13D11 directed against Vma2p used at 1:1000 (Molecular Probes), or the monoclonal antibody 10E5 used to probe for CPY at 1:500 (Molecular Probes). Proteins were visualized using affinity-purified donkey anti-mouse secondary antibodies conjugated to horseradish peroxidase (1:20000) (Jackson ImmunoResearch Laboratories Inc.) and chemiluminescence (Amersham Biosciences) Fluorescence Microscopy—Quinacrine staining of live yeast cells was conducted as previously described (36Conibear E. Stevens T.H. Methods Enzymol. 2002; 351: 408-432Crossref PubMed Scopus (45) Google Scholar) with the following modification. Concanavalin A TRITC (Molecular Probes) was added at a final concentration of 50 μg/ml with the quinacrine to allow for visualization of the cell surface. Images were acquired on a Zeiss Axioplan 2 microscope and manipulated using AxioVision software. Native Immunoprecipitation—Immunoprecipitations were carried out as previously described with the following exceptions (16Powell B. Graham L.A. Stevens T.H. J. Biol. Chem. 2000; 275: 23654-23660Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). 1 mg of solubilized membranes was precleared with the addition of 50 μl of 50% slurry of Protein G-Sepharose 4 Fast Flow (Amersham Biosciences). 5 μl of affinity-purified goat anti-HA antibody (Novus Biologicals) was added to the pre-cleared supernatant and agitated at 4 °C for 1 h. 50 μl of 50% slurry of protein G-Sepharose 4 fast flow was added and incubated at 4 °C for 1 h. The beads were collected with a brief centrifugation and washed 3 times with ice-cold phosphate-buffered saline plus 1% C12E9. The beads were resuspended in 200 μl of Thorner buffer, and 20 μl of the supernatant was loaded per well of SDS-PAGE and analyzed by immunoblot analysis. V-ATPase Assays—ATPase activity was measured by a coupled spectrophotometric assay in the absence and presence of 1 μm concanamycin A, as described previously (36Conibear E. Stevens T.H. Methods Enzymol. 2002; 351: 408-432Crossref PubMed Scopus (45) Google Scholar). Determining Topology of C Termini for Vma3p, Vma11p, and Vma16p—To determine whether the C termini of Vma3p, Vma11p, and Vma16p are located on the cytosolic face of the vacuole or in the lumen, the proteins were epitope-tagged with the HA epitope on their C termini. Previous reports demonstrated that strains expressing tagged copies of Vma3p, Vma11p, or Vma16p produced V-ATPase complexes indistinguishable from wild-type cells (15Hirata R. Graham L.A. Takatsuki A. Stevens T.H. Anraku Y. J. Biol. Chem. 1997; 272: 4795-4803Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 16Powell B. Graham L.A. Stevens T.H. J. Biol. Chem. 2000; 275: 23654-23660Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), indicating that the epitope-tagged forms of these proteins are fully functional. To determine the topology of the proteins, the intact vesicles were isolated from strains expressing tagged Vma3p, Vma11p, or Vma16p and subjected to limited proteolysis. Vacuoles were incubated with a mixture of proteinase K and trypsin either in the presence or absence of 1% Triton X-100 at 0 °C for 10 min (21Jackson D.D. Stevens T.H. J. Biol. Chem. 1997; 272: 25928-25934Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). SDS-PAGE and immunoblot analysis were performed on the time points taken during the protease reaction. Two proteins, Vph1p and CPY, were monitored as controls. Vph1p confirms the accessibility of the proteases to proteins oriented on the cytoplasmic side, whereas CPY validates the intactness of the vacuoles. Using the 7B1 anti-Vph1p antibody, Jackson and Stevens (21Jackson D.D. Stevens T.H. J. Biol. Chem. 1997; 272: 25928-25934Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) show that the N-terminal domain of Vph1p is located in the cytoplasm and is easily susceptible to proteolytic degradation without compromising the integrity of the membrane. CPY is translated with a propeptide domain, which is cleaved when CPY reaches the vacuole to yield the mature form of CPY (36Conibear E. Stevens T.H. Methods Enzymol. 2002; 351: 408-432Crossref PubMed Scopus (45) Google Scholar). Because vacuoles were isolated from a yeast strain deficient in active vacuolar proteases due to a mutation in the PEP4 gene, only the proCPY form was present in the vacuole. The anti-Vph1p blot in Fig. 1a illustrates that the protein is degraded by protease in both the absence or presence of detergent, confirming the proteases were active. In contrast, the shift of the proCPY form to the mature CPY can only be seen in the presence of detergent, confirming the intactness of the vacuoles. These results indicate that the isolated vacuoles were properly oriented and intact throughout the limited proteolysis assay. The samples were also probed using an antibody directed against the HA epitope (Fig. 1a). The epitopes present on Vma3p, Vma11p, and Vma16p were intact after 10-min of incubation with the proteases in the absence of detergent, indicating that they were not accessible to the proteases. The epitopes were only digested when the membranes were solubilized before the addition of the proteases. These results are consistent with the C termini of Vma3p, Vma11p, and Vma16p oriented toward the lumen of the vacuole. The N Termini of Vma3p and Vma11p Are Lumenal—Hydropathy plots predict that Vma3p and Vma11p contain four transmembrane segments. Therefore, if this model is correct and the C termini are in the lumen of the vacuole, then the N termini should also be present within the lumen. To test this hypothesis, N-terminal epitope-tagged Vma3p and Vma11p were constructed. Yeast deletion strains of vma3Δ or vma11Δ expressing the corresponding plasmid-borne N-terminal tagged protein grew on pH 7.5-buffered media containing 100 mm CaCl2 (data not shown), a standard test for V-ATPase function (3Graham L.A. Flannery A.R. Stevens T.H. J. Bioenerg. Biomembr. 2003; 35: 301-312Crossref PubMed Scopus (84) Google Scholar). Vacuoles were isolated from cells expressing N-terminal-tagged Vma3p or Vma11p and also subjected to protease digestion followed by immunoblot analysis. As with the C-terminal epitopes, Fig. 1b shows that the N-terminal HA epitope of both Vma3p and Vma11p were protected from proteolysis in the absence of detergent and degraded only with detergent present. These results support the hypothesis that there are four transmembrane regions and the N termini, and C termini of both Vma3p and Vma11p are orientated toward the lumen of the vacuole. The N Terminus of Vma16p Is Cytosolic—Unlike Vma3p or Vma11p, Vma16p is predicted to form five putative transmembrane regions. Nishi et al. (33Nishi T. Kawasaki-Nishi S. Forgac M. J. Biol. Chem. 2003; 278: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 38Nishi T. Kawasaki-Nishi S. Forgac M. J. Biol. Chem. 2001; 276: 34122-34130Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) suggest that the N termini and C termini of Vma16p are both cytosolic, implying that there would be only four transmembrane regions. However, other studies suggest that Vma16p has five transmembrane regions (34Gibson L.C. Cadwallader G. Finbow M.E. Biochem. J. 2002; 366: 911-919Crossref PubMed Google Scholar, 39Kim H. Melen K. von Heijne G. J. Biol. Chem. 2003; 278: 10208-10213Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Our results indicate that the C terminus of Vma16p is oriented toward the lumen (Fig. 1a). If Vma16p forms five transmembrane regions, then one would predict the N terminus to be oriented toward the cytosol, whereas if only four transmembrane regions are present, then the N terminus of Vma16p should be located in the lumen of the vacuole. To further test these models a doubly epitope-tagged protein was constructed, subjected to protease treatment, and analyzed by immunoblot analysis. The c-Myc epitope was placed on the N terminus of the previously C-terminal HA-tagged Vma16p. Cells expressing a copy of the doubly epitope-tagged Vma16p were able to confer wild-type growth on pH 7.5 YEPD containing 100 mm CaCl2 (Fig. 2c). Vacuoles were isolated from cells expressing the doubly tagged Vma16p and treated with proteases in the presence or absence of detergent. The Vph1p and CPY controls indicated that the proteases were active, and the vacuoles were intact (Fig. 2a). Samples probed with an antibody recognizing the c-Myc tag illustrate that the Myc epitope is labile both in the absence and presence of detergent, indicating that Vma16p contains five transmembrane regions since the N terminus of Vma16p is on the cytosolic face of the vacuole (Fig. 2a). The C-terminal HA tag was protected from protease digestion unless the membranes were solubilized with detergent as previously observed (Fig. 2a). The same results were observed for the N terminus and the C terminus of Vma16p if the epitope tags were transposed (N-terminal HA and C-terminal c-Myc; data not shown). These results support the model th" @default.
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• W2083872341 date "2004-09-01" @default.
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• W2083872341 title "Topological Characterization of the c, c′, and c″ Subunits of the Vacuolar ATPase from the Yeast Saccharomyces cerevisiae" @default.
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• W2083872341 doi "https://doi.org/10.1074/jbc.m406767200" @default.
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