TM2 but Not TM4 of Subunit c″ Interacts with TM7 of Subunit a of the Yeast V-ATPase as Defined by Disulfide-mediated Cross-linking
2004; Elsevier BV; Volume: 279; Issue: 43 Linguagem: Inglês
10.1074/jbc.m407345200
ISSN1083-351X
AutoresYanru Wang, Takao Inoué, Michael Forgac,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoThe vacuolar (H+)-ATPase (or V-ATPase) is an ATP-dependent proton pump which couples the energy released upon ATP hydrolysis to rotational movement of a ring of proteolipid subunits (c, c′, and c″) relative to the integral subunit a. The proteolipid subunits each contain a single buried acidic residue that is essential for proton transport, with this residue located in TM4 of subunits c and c′ and TM2 of subunit c″. Subunit c″ contains an additional buried acidic residue in TM4 that is not required for proton transport. The buried acidic residues of the proteolipid subunits are believed to interact with an essential arginine residue (Arg735) in TM7 of subunit a during proton translocation. We have previously shown that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM4 of subunit c′ bordered by Glu145 and Leu147 (Kawasaki-Nishi et al. (2003) J. Biol. Chem. 278, 41908–41913). We have now analyzed interaction of subunits a and c″ using disulfide-mediated cross-linking. The results indicate that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM2 of subunit c″ centered on Ile105, with the essential glutamic acid residue (Glu108) located near the opposite border of this face compared with TM4 of subunit c′. By contrast, TM4 of subunit c″ does not form strong cross-links with TM7 of subunit a, suggesting that these transmembrane segments are not normally in close proximity. These results are discussed in terms of a model involving rotation of interacting helices in subunit a and the proteolipid subunits relative to each other. The vacuolar (H+)-ATPase (or V-ATPase) is an ATP-dependent proton pump which couples the energy released upon ATP hydrolysis to rotational movement of a ring of proteolipid subunits (c, c′, and c″) relative to the integral subunit a. The proteolipid subunits each contain a single buried acidic residue that is essential for proton transport, with this residue located in TM4 of subunits c and c′ and TM2 of subunit c″. Subunit c″ contains an additional buried acidic residue in TM4 that is not required for proton transport. The buried acidic residues of the proteolipid subunits are believed to interact with an essential arginine residue (Arg735) in TM7 of subunit a during proton translocation. We have previously shown that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM4 of subunit c′ bordered by Glu145 and Leu147 (Kawasaki-Nishi et al. (2003) J. Biol. Chem. 278, 41908–41913). We have now analyzed interaction of subunits a and c″ using disulfide-mediated cross-linking. The results indicate that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM2 of subunit c″ centered on Ile105, with the essential glutamic acid residue (Glu108) located near the opposite border of this face compared with TM4 of subunit c′. By contrast, TM4 of subunit c″ does not form strong cross-links with TM7 of subunit a, suggesting that these transmembrane segments are not normally in close proximity. These results are discussed in terms of a model involving rotation of interacting helices in subunit a and the proteolipid subunits relative to each other. The vacuolar (H+)-ATPases (or V-ATPases) 1The abbreviations used are: V-ATPase, vacuolar proton-translocating adenosine triphosphatase; F-ATPase, F1F0 ATP synthase; HA, influenza hemagglutinin; TM, transmembrane segment; ACMA, 9-amino-6-chloro-2-methoxyacridine; Mes, 4-morpholineethanesulfonic acid.1The abbreviations used are: V-ATPase, vacuolar proton-translocating adenosine triphosphatase; F-ATPase, F1F0 ATP synthase; HA, influenza hemagglutinin; TM, transmembrane segment; ACMA, 9-amino-6-chloro-2-methoxyacridine; Mes, 4-morpholineethanesulfonic acid. are a family of ATP-dependent proton pumps that function in both intracellular compartments and the plasma membrane (1Nishi T. Forgac M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 94-103Crossref PubMed Scopus (985) Google Scholar, 2Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 3Graham L.A. Powell B. Stevens T.H. J. Exp. Biol. 2000; 203: 61-70Crossref PubMed Google Scholar, 4Kane P.M. Parra K.J. J. Exp. Biol. 2000; 203: 81-87Crossref PubMed Google Scholar, 5Bowman E.J. Bowman B.J. J. Exp. Biol. 2000; 203: 97-106Crossref PubMed Google Scholar, 6Nelson N. Perzov N. Cohen A. Hagai K. Padler V. Nelson H. J. Exp. Biol. 2000; 203: 89-95Crossref PubMed Google Scholar, 7Futai M. Oka T. Sun-Wada G. Moriyama Y. Kanazawa H. Wada Y. J. Exp. Biol. 2000; 203: 107-116Crossref PubMed Google Scholar, 8Sze H. Li X. Palmgren M.G. Plant Cell. 1999; 11: 677-690PubMed Google Scholar). Within intracellular compartments such as lysosomes, endosomes, the Golgi and secretory vesicles, V-ATPases function in a variety of processes, including protein degradation, receptor-mediated endocytosis, viral entry, intracellular membrane traffic, protein processing, and neurotransmitter uptake (1Nishi T. Forgac M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 94-103Crossref PubMed Scopus (985) Google Scholar). Plasma membrane V-ATPases have been shown to function in acid secretion in the kidney, bone degradation by osteoclasts, pH homeostasis in macrophages and neutrophils, sperm maturation in the vas deferens, K+ transport by insect goblet cells, and invasion by tumor cells (9Brown D. Breton S. J. Exp. Biol. 2000; 203: 137-145Crossref PubMed Google Scholar, 10Li Y.-P. Chen W. Liang Y. Li E. Stashenko P. Nat. Genet. 1999; 23: 447-451Crossref PubMed Scopus (416) Google Scholar, 11Nanda A. Brumell J.H. Nordström T. Kjeldsen L. Sengelov H. Borregaard N. Rotstein O.D. Grinstein S. J. Biol. Chem. 1996; 271: 15963-15970Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 12Wieczorek H. Grüber G. Harvey W.R. Huss M. Merzendorfer H. Zeiske W. J. Exp. Biol. 2000; 203: 127-135Crossref PubMed Google Scholar, 13Sennoune S.R. Bakunts K. Martinez G.M. Chua-Tuan J.L. Kebir Y. Attaya M.N. Martinez-Zaguilan R. Am. J. Physiol. 2004; 286: C1443-C1452Crossref PubMed Scopus (283) Google Scholar).Subunits of the V-ATPase are organized into a peripheral domain (V1) responsible for ATP hydrolysis and an integral domain (V0) that carries out proton translocation (1Nishi T. Forgac M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 94-103Crossref PubMed Scopus (985) Google Scholar, 2Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 3Graham L.A. Powell B. Stevens T.H. J. Exp. Biol. 2000; 203: 61-70Crossref PubMed Google Scholar, 4Kane P.M. Parra K.J. J. Exp. Biol. 2000; 203: 81-87Crossref PubMed Google Scholar, 5Bowman E.J. Bowman B.J. J. Exp. Biol. 2000; 203: 97-106Crossref PubMed Google Scholar, 6Nelson N. Perzov N. Cohen A. Hagai K. Padler V. Nelson H. J. Exp. Biol. 2000; 203: 89-95Crossref PubMed Google Scholar, 7Futai M. Oka T. Sun-Wada G. Moriyama Y. Kanazawa H. Wada Y. J. Exp. Biol. 2000; 203: 107-116Crossref PubMed Google Scholar, 8Sze H. Li X. Palmgren M.G. Plant Cell. 1999; 11: 677-690PubMed Google Scholar). ATP hydrolysis in the V1 domain occurs at nucleotide binding sites located at the interface of the A and B subunits (14Liu Q. Kane P.M. Newman P.R. Forgac M. J. Biol. Chem. 1996; 271: 2018-2022Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 15MacLeod K.J. Vasilyeva E. Baleja J.D. Forgac M. J. Biol. Chem. 1998; 273: 150-156Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), which form the hexameric structure observed in electron microscopic images of the V-ATPase (16Gruber G. Radermacher M. Ruiz T. Godovac-Zimmermann J. Canas B. Kleine-Kohlbrecher D. Huss M. Harvey W.R. Wieczorek H. Biochemistry. 2000; 39: 8609-8616Crossref PubMed Scopus (69) Google Scholar). ATP hydrolysis has been shown to drive rotation of a central stalk, composed of the D and F subunits (17Imamura 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 (163) Google Scholar, 18Arata Y. Baleja J.D. Forgac M. Biochemistry. 2002; 41: 11301-11307Crossref PubMed Scopus (77) Google Scholar), which in turn drives rotation of a ring of proteolipid subunits (c, c′, and c″) relative to subunit a (19Hirata 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 (146) Google Scholar, 20Yokoyama K. Nakano M. Imamura H. Yoshida M. Tamakoshi M. J. Biol. Chem. 2003; 278: 24255-24258Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Subunit a is an integral membrane protein possessing an N-terminal hydrophilic domain located on the cytoplasmic side of the membrane and a hydrophobic C-terminal domain containing nine transmembrane segments (21Leng X.H. Nishi T. Forgac M. J. Biol. Chem. 1999; 274: 14655-14661Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Subunit a is held fixed relative to the hydrolytic head of V1 by a peripheral stalk composed of subunits C, E, G, H, and the hydrophilic domain of subunit a (18Arata Y. Baleja J.D. Forgac M. Biochemistry. 2002; 41: 11301-11307Crossref PubMed Scopus (77) Google Scholar, 22Arata Y. Baleja J.D. Forgac M. J. Biol. Chem. 2002; 277: 3357-3363Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 23Landolt-Marticorena C. Williams K.M. Correa J. Chen W. Manolson M.F. J. Biol. Chem. 2000; 275: 15449-15457Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar).The membrane integral domain of subunit a contains a number of buried charged residues, including Glu789, His743, and Arg799, whose mutation results in partial inhibition of proton transport (24Leng X.H. Manolson M. Liu Q. Forgac M. J. Biol. Chem. 1996; 271: 22487-22493Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 25Leng X.H. Manolson M. Forgac M. J. Biol. Chem. 1998; 273: 6717-6723Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 26Kawasaki-Nishi S. Nishi T. Forgac M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12397-12402Crossref PubMed Scopus (116) Google Scholar). The only a subunit residue absolutely required for proton translocation is Arg735 located in TM7 (26Kawasaki-Nishi S. Nishi T. Forgac M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12397-12402Crossref PubMed Scopus (116) Google Scholar). Even conservative replacement of this residue with lysine results in complete loss of proton transport (26Kawasaki-Nishi S. Nishi T. Forgac M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12397-12402Crossref PubMed Scopus (116) Google Scholar). Arg735 has been postulated to function in displacement of protons bound to buried acidic residues on the ring of proteolipid subunits, analogous to the function of Arg210 in proton transport by the F-ATPases (27Vik S.B. Long J.C. Wada T. Zhang D. Biochim. Biophys. Acta. 2000; 1458: 457-466Crossref PubMed Scopus (67) Google Scholar, 28Cain B.D. J. Bioenerg. Biomemb. 2000; 32: 365-371Crossref PubMed Scopus (59) Google Scholar, 29Fillingame R.H. Angevine C.M. Dmitriev O.Y. Biochim. Biophys. Acta. 2002; 1555: 29-36Crossref PubMed Scopus (73) Google Scholar).The V-ATPases contain three different proteolipid subunits (c, c′, and c″) which are present in a stoichiometry of one copy each of c′ and c″ and 4–5 copies of c (30Arai H. Terres G. Pink S. Forgac M. J. Biol. Chem. 1988; 263: 8796-8802Abstract Full Text PDF PubMed Google Scholar, 31Powell B. Graham L.A. Stevens T.H. J. Biol. Chem. 2000; 275: 23654-23660Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Subunits c and c′ are composed of four transmembrane segments (32Mandel 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 (238) Google Scholar, 33Hirata 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) whereas subunit c″ contains four or five transmembrane segments (34Nishi T. Kawasaki-Nishi S. Forgac M. J. Biol. Chem. 2003; 278: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 35Flannery A.R. Graham L.A. Stevens T.H. J. Biol. Chem. 2004; 279: 39856-39862Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Each proteolipid subunit contains a single glutamic acid residue buried in the middle of one of these segments that is essential for proton transport (33Hirata 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). For subunits c and c′, the essential glutamic acid residue is present in the last transmembrane segment, whereas for subunit c″ the essential glutamic acid residue is present near the middle of the molecule (32Mandel 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 (238) Google Scholar, 33Hirata 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, 34Nishi T. Kawasaki-Nishi S. Forgac M. J. Biol. Chem. 2003; 278: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 35Flannery A.R. Graham L.A. Stevens T.H. J. Biol. Chem. 2004; 279: 39856-39862Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). It is these essential glutamate residues that are believed to undergo reversible protonation and deprotonation during rotary catalysis and that are thought to interact with Arg735 of subunit a to activate proton release. It is therefore important to define the helical interactions that occur between the proteolipid subunits and subunit a within the V0 domain.We have previously demonstrated by cysteine-mediated cross-linking that TM7 of subunit a is in close proximity to TM4 of subunit c′ and have identified the helical faces of these subunits that interact (36Kawasaki-Nishi S. Nishi T. Forgac M. J. Biol. Chem. 2003; 278: 41908-41913Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In the present study we have extended this analysis to the interaction of subunit a and subunit c″, whose topology and location of essential residues make it unique among the proteolipid subunits of both the V and F-ATPases.EXPERIMENTAL PROCEDURESMaterials and Strains—Zymolyase 100T was obtained from Seikagaku America, Inc. Protease inhibitors and the monoclonal antibody 3F10 (directed against the HA antigen) that is conjugated with horseradish peroxidase were from Roche Applied Science. The monoclonal antibody 10D7 against the yeast 100 kDa a subunit Vph1p (37Kane P.M. Kuehn M.C. Howald-Stevenson I. Stevens T. J. Biol. Chem. 1992; 267: 447-454Abstract Full Text PDF PubMed Google Scholar) was from Molecular Probes. Escherichia coli and yeast culture media were purchased from Difco Laboratories. Restriction endonucleases, T4 DNA ligase and other molecular biology reagents were from Fisher and New England Biolabs. Phenylmethylsulfonyl fluoride and most other chemicals were purchased from Sigma. Yeast strains lacking the VMA16, VPH1, and STV1 genes were constructed by replacing the entire coding region of VMA16 with the TRP gene and insertion of the LEU gene into the VPH1 gene and the LYS gene STV1 gene at the positions indicated by Manolson et al. (38Manolson 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) using the YPH500 strain (MAT alpha ade2, ura3, leu2, his3, trp1, lys2). The proteolipid Vma16p was tagged at the C terminus with the 9-amino acid epitope (YPYDVPDYA) from influenza hemagglutinin (HA) as described previously (34Nishi T. Kawasaki-Nishi S. Forgac M. J. Biol. Chem. 2003; 278: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). YEPD buffered to pH 5.5 or pH 7.5 was used for selection of strains showing a vma– phenotype.Transformation and Selection—Site-directed mutants of Vph1p and Vma16p were constructed using the Altered Sites II in vitro mutagenesis system (Promega), and the presence of the mutations was verified by sequencing the entire length of subcloned DNA. Plasmids carrying mutations of a and c″ are co-transformed into yeast cells lacking functional endogenous Vph1p, Stv1p, and Vma16p by the lithium acetate method (39Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2877) Google Scholar). The transformants were selected on histidine minus and uracil minus plates and growth phenotypes of the mutants were assessed on YEPD plates buffered with 50 mm KH2PO4 or 50 mm succinic acid to either pH 7.5 or pH 5.5.Analysis of Subunit Expression and V-ATPase Assembly—Yeast vacuolar membranes and whole cell lysates were prepared using the protocol described previously (40Horazdovsky B.F. Emr S.D. J. Biol. Chem. 1993; 268: 4953-4962Abstract Full Text PDF PubMed Google Scholar, 41Graham L.A. Hill K.J. Stevens T.H. J. Cell Biol. 1998; 142: 39-49Crossref PubMed Scopus (80) Google Scholar). Whole cell lysates and vacuolar membrane enriched fractions were separated by SDS-PAGE on 4–15% gradient acrylamide gels. The presence of Vma16p on vacuolar membranes was detected by Western blotting using a horseradish peroxidase-conjugated monoclonal antibody 3F10 against HA, while Vph1p was detected using the monoclonal antibody 10D7 (Molecular Probes), followed by a horseradish peroxidase-conjugated secondary antibody (Bio-Rad) (36Kawasaki-Nishi S. Nishi T. Forgac M. J. Biol. Chem. 2003; 278: 41908-41913Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Blots were developed using a chemiluminescence detection method obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD).Cross-linking of Subunits a and c″ by Cu(1,10-phenanthroline)2SO4 (CuP)—Cross-linking between cysteine residues introduced into subunits a and c″ was performed using the protocol described previously by Jiang and Fillingame (42Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Crossref PubMed Scopus (150) Google Scholar). Vacuolar membrane vesicles (∼50μg of protein) were washed in labeling buffer (5 mm Tris-Mes, pH 7.5, 0.25 mm MgCl2 and 1.1 m glycerol). 2.5 mm Cu(1,10-phenanthroline)2SO4 was added, and samples were incubated for 60 min at room temperature to catalyze disulfide bond formation. The reaction was terminated by addition of EDTA and N-ethylmaleimide to a final concentration of 15 mm and 20 mm, respectively. Samples were then subjected to SDS-PAGE on 4–15% acrylamide gels and transferred to nitrocellulose membranes. The blots were probed with horseradish peroxidase-conjugated monoclonal antibody 3F10 against the HA epitope tag (36Kawasaki-Nishi S. Nishi T. Forgac M. J. Biol. Chem. 2003; 278: 41908-41913Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Blots were developed using the Supersignal ULTRA chemiluminescent system (Pierce).Other Procedures—ATPase activity was measured using a coupled spectrophotometric assay and ATP-dependent proton transport was measured by fluorescence quenching using the fluorescence probe ACMA as described previously (43Feng Y. Forgac M. J. Biol. Chem. 1992; 267: 5817-5822Abstract Full Text PDF PubMed Google Scholar). All assays were carried out in the presence or absence of 1 μm concanamycin A, a specific inhibitor of the V-ATPase (44Drose S. Bindseil K.U. Bowman E.J. Siebers A. Zeeck A. Altendorf K. Biochemistry. 1993; 32: 3902-3906Crossref PubMed Scopus (369) Google Scholar). SDS-polyacrylamide gel electrophoresis was carried out as described by Laemmli (45Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205955) Google Scholar).RESULTSGrowth Phenotype of Yeast Strains Expressing Single Cysteine-containing Mutant Forms of Vma16p and Vph1p—To study the interaction between subunits a and c″, a yeast strain disrupted in the two genes that encode subunit a (VPH1 and STV1) as well as the gene encoding subunit c″ (VMA16) was constructed as follows. Using the parental strain YPH500, the VMA16 gene was replaced with the TRP gene, and the LEU and LYS genes were inserted into the VPH1 and STV1 genes, respectively, as previously described (38Manolson 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, 46Manolson M.F. Proteau D. Preston R.A. Stenbit A. Roberts B.T. Hoyt M. Preuss D. Mulholland J. Botstein D. Jones E.W. J. Biol. Chem. 1992; 267: 14294-14303Abstract Full Text PDF PubMed Google Scholar). The VMA16 gene was expressed in this strain using the pRS413 plasmid containing the HIS marker whereas the VPH1 gene was expressed using the pRS316 plasmid containing the URA marker. A Cys-less form of Vph1p has previously been shown to support wild-type growth at pH 7.5 (21Leng X.H. Nishi T. Forgac M. J. Biol. Chem. 1999; 274: 14655-14661Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), suggesting it forms a V-ATPase complex possessing at least 20% of wild-type levels of activity in vitro (47Liu J. Kane P.M. Biochemistry. 1996; 35: 10938-10948Crossref PubMed Scopus (55) Google Scholar, 15MacLeod K.J. Vasilyeva E. Baleja J.D. Forgac M. J. Biol. Chem. 1998; 273: 150-156Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Cells co-expressing Cys-less forms of both Vph1p and Vma16p also displayed wild-type growth at pH 7.5, indicating that none of the endogenous cysteine residues in either protein are essential for activity.TM7 of Vph1p contains a buried arginine residue (Arg735) that is absolutely required for proton transport by the V-ATPase and that has been proposed to interact with the buried carboxyl groups on the proteolipid subunits (26Kawasaki-Nishi S. Nishi T. Forgac M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12397-12402Crossref PubMed Scopus (116) Google Scholar). Unique cysteine residues were therefore introduced at nine different positions along TM7 of Vph1p, including sites predicted to reside on the same helical face as Arg735 (Ser728, Ala731, Ser732, Ala738, Leu739, and Ala742), sites adjoining this helical face (Leu734 and Leu736) and sites on the opposite helical face (Tyr733). Unique cysteine residues were also introduced into the two transmembrane helices of Vma16p that contain buried acidic residues. Assuming a four transmembrane segment model of Vma16p (34Nishi T. Kawasaki-Nishi S. Forgac M. J. Biol. Chem. 2003; 278: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), these correspond to TM2 and TM4 (see “Discussion”). In TM2 of Vma16p, which contains the essential glutamic acid residue Glu108, cysteine residues were introduced at Ser103, Ile104, Ile105, Phe106, Ser107, Glu108, Val109, Val110, Ala111, and Ile112. These residues correspond to approximately the central half of TM2 of Vma16p and represent all of the helical faces of this segment (33Hirata 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). In TM4 of Vma16p, which contains a buried glutamic acid reside (Glu188) that is not essential for proton transport (33Hirata 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), cysteine residues were introduced at Ile184, Leu185, Val186, Ile187, Glu188, Ile189, Phe190, Gly191, Ser192, and Ile193. The yeast strain disrupted in VMA16, VPH1, and STV1 was then transformed with plasmids bearing the Cys-less form or one of the single cysteine-containing mutants of Vma16p as well as one of the single cysteine-containing mutants of Vph1p. The growth phenotype of the resultant 189 double-replacement strains at pH 7.5 is shown in Table I. As expected from previous results, strains bearing mutations at Glu108 of Vma16p showed no growth at neutral pH whereas most strains expressing the E188C mutant of Vma16p displayed a wild-type growth phenotype (33Hirata 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). For mutations in the a subunit, the only mutation to seriously compromise growth at pH 7.5 was Y733C. The majority of the 189 double mutants tested showed normal (or near normal) growth at neutral pH, suggesting that the resultant V-ATPase complexes possessed significant activity in vivo.Table IGrowth phenotypes of Cys-substituted mutants at pH 7.5Subunit c″TM7 of subunit aSer728Ala731Ser732Tyr733Leu734Leu736Ala738Leu739Ala742Cys-less+++a+++ indicates wild-type growth.+++++b++ indicates partially defective growth.+c+ indicates severely defective growth.+++++++++++++++Ser103++++++++++++++++++++++Ile104+++++++++++++++++++++++Ile105+++++++++++++++++++Phe106++++++++++++++++++++++++Ser107++++++++++++++++++++++++Glu108-d- indicates no growth.--------Val109+++++++++++++++++++++++Val110++++++++++++++++++++++++Ala111++++++++++++++++++++++++Ile112++++++++++++++++++++++++Ile184++++++++++++++++++++++++Leu185++++++++++++++++++++++++Val186+++++++++++++++++++++++Ile187+++++++++++++++++++++++Glu188+++++++++++++++++++++++Ile189++++++++++++++++++++++++Phe190+++++++-++++++++++++++Gly191+++++++++++++++++++++++Ser192+++++++++++++++++++++++Ile193++++++++++++++++++++a +++ indicates wild-type growth.b ++ indicates partially defective growth.c + indicates severely defective growth.d - indicates no growth. Open table in a new tab Assembly of V0 Complexes Containing Cysteine Mutations in Subunits a and c″—To assess the effects of the mutations in Vma16p and Vph1p on assembly of the V0 domain, partially purified vacuolar membranes were subjected to SDS-PAGE and Western blot analysis was performed using antibodies against both Vph1p and the HA-tagged Vma16p. As can be seen in Fig. 1, vacuolar membranes from all of the double replacement strains tested showed wild-type levels of both Vph1p and Vma16p, suggesting normal assembly and targeting of the V0 domain (37Kane P.M. Kuehn M.C. Howald-Stevenson I. Stevens T. J. Biol. Chem. 1992; 267: 447-454Abstract Full Text PDF PubMed Google Scholar).While proton transport and ATPase activities have not been measured for vacuoles from the 189 double mutant strains analyzed, it was felt that useful information about the arrangement of V0 subunits could be obtained even from complexes lacking substantial activity, as for those displaying a vma– phenotype (Table I). This is similar to previous studies of contacts between subunits a and c′ of the V-ATPase (36Kawasaki-Nishi S. Nishi T. Forgac M. J. Biol. Chem. 2003; 278: 41908-41913Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) and subunits a and c of the F-ATPase (42Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Crossref PubMed Scopus (150) Google Scholar), where many of the strongest cross-links were observed for complexes lacking activity as assessed by their growth phenotype.ATPase and Proton Transport Activity of E108Q and E188Q Mutants of Subunit c″—It had previously been reported that the mutations E108Q in TM2 but not E188Q in TM4 of subunit c″ led to a vma–phenotype (33Hirata 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). In addition, the E108Q mutation resulted in loss of 99% of wild-type ATPase activity in isolated vacuolar membranes and qualitative loss of vacuolar acidification as assessed by quinacrine staining of vacuoles in vivo (33Hirata 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). By contrast, the effect of the E188Q mutation on ATPase activity was not determined and the effect of this mutation on proton transport was not quantitated. We therefore measured both proton transport and concanamycin-sensitive ATPase activity in vacuolar membranes isolated from the wild-type strain and strains expressing the E108Q or E188Q mutations in subunit c″ as described under “Experimental Procedures.” In agreement with the previous findings (33Hirata 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), vacuolar membranes from the strain expressing the E108Q mutant of subunit c″ possessed no detectable ATP-dependent proton transport or concanamycin-sensitive ATPase activity. Interestingly, vacuolar membranes from the strain expressing the E188Q mutant had 87 ± 7% of the wild-type ATPase activity but only 64 ± 4% of wild-type proton transport activity. These results suggest a partial uncoupling of the E188Q mutant and a possible function of this residue in proton transport.Cysteine-mediated Cross-linking of Subunits a and c″ Using Cupric Phenanthroline—To determine the proximity of cysteine residues introduced into TM7 of subunit a to cysteine residues introduced into TM2 and TM4 of subunit c″, vacuolar membranes were isolated from each of the strains expressing single cysteine-containing forms of Vph1p and Vma16p followed by cross-linking using cupric phenanthroline, as described under “Experimental Procedures.” Samples were separated by SDS-PAGE and Western blotting was performed using the horseradish peroxidase-conjugated monoclonal antibody 3F10 directed against the HA epitope tag introduced at the C terminus of Vma16p. Cross-linking was tested for all 189 strains expressing single cysteine-containing f
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