Stoichiometry of the Peripheral Stalk Subunits E and G of Yeast V1-ATPase Determined by Mass Spectrometry
2007; Elsevier BV; Volume: 283; Issue: 6 Linguagem: Inglês
10.1074/jbc.m707924200
ISSN1083-351X
AutoresNorton Kitagawa, Hortense Mazon, Albert J. R. Heck, Stephan Wilkens,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe stoichiometry of yeast V1-ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V1-ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V1-ATPase purified from yeast grown on 14N-containing medium were titrated with defined amounts of 15N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, 14N- and 15N-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V1-ATPase is A3B3DE3FG3H. The stoichiometry of yeast V1-ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V1-ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V1-ATPase purified from yeast grown on 14N-containing medium were titrated with defined amounts of 15N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, 14N- and 15N-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V1-ATPase is A3B3DE3FG3H. Vacuolar ATPases (V-ATPases, 3The abbreviations used are: V-ATPasevacuolar ATPaseV1V0proton-pumping vacuolar ATPaseV1water soluble domain of the vacuolar proton-pumping ATPaseV0membrane-bound domain of the proton-pumping vacuolar ATPaseMSmass spectrometryMALDImatrix assisted laser desorption ionizationTOFtime-of-flightCVcolumn volumesMBPmaltose-binding protein. V1V0-ATPases) are ATP hydrolysis-driven proton pumps found in the endomembrane system of eukaryotic organisms, where they function to acidify the interior of subcellular organelles such as lysosomes, early and late endosomes, clathrin-coated vesicles, the Golgi, the plant tonoplast, and the yeast vacuole (1Kane P.M. Microbiol. Mol. Biol. Rev. 2006; 70: 177-191Crossref PubMed Scopus (330) Google Scholar, 2Nishi T. Forgac M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 94-103Crossref PubMed Scopus (1005) Google Scholar, 3Nelson N. Harvey W.R. Physiol. Rev. 1999; 79: 361-385Crossref PubMed Scopus (371) Google Scholar, 4Finbow M.E. Harrison M.A. Biochem. J. 1997; 324: 697-712Crossref PubMed Scopus (234) Google Scholar). In higher organisms, the V-ATPase complex can also be found in the plasma membrane of polarized cells involved in acid secretion such as the ruffled membrane of bone osteoclasts or the apical membrane of renal intercalated cells. The vacuolar ATPase is a large, multisubunit complex, which can be divided into a water-soluble ATPase domain and a membrane-bound proton pore. The two domains are termed V1 and V0, respectively, in analogy to the F1 and F0 of the related F1F0-ATP synthase. In yeast, the V1-ATPase domain contains subunits AB(C)DEFGH, whereas the membrane-bound V0 is made of subunits acc′c″de. Much like the F-ATP synthase, the V-ATPase is a rotary molecular motor enzyme (5Hirata T. Iwamoto-Kihara A. Sun-Wada G-H. Okajima T. Wada Y. Futai M. J. Biol. Chem. 2003; 277: 23714-23719Abstract Full Text Full Text PDF Scopus (148) Google Scholar, 6Imamura H. Nakano M. Noji H. Muneyuki E. Ohkuma S. Yoshida M. Yokoyama K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2313-2315Google Scholar); ATP hydrolysis taking place on the A subunits of the A3B3 catalytic domain is coupled to proton translocation across the membrane domain via rotation of a central stalk made of subunits D, F, and d and a proteolipid ring (subunits c, c′, and c″). The remaining subunits C, E, G, and H are involved in forming a peripheral stator domain that provides a structural link between the catalytic domain (A3B3) and the membrane-bound a subunit. In the related F-ATP synthase, it is now well established that there is a single peripheral stalk, which, in the case of the bacterial enzyme, is formed by two copies of the membrane-anchored b subunits and the δ subunit (7Dunn S.D. McLachlin D.T. Revington M. Biochim. Biophys. Acta. 2000; 1458: 356-363Crossref PubMed Scopus (82) Google Scholar). The situation in the vacuolar ATPase, however, is more complicated in that there appear to be multiple peripheral stalks that connect the catalytic domain to the membrane-bound a subunit, possibly via the V-ATPase-specific H and C subunits. Using electron microscopy and single particle image analysis, we have previously shown that the C and H subunits are positioned in the interface connecting the V1 and V0, where they are connected to the A3B3 domain via elongated protein densities bound at the periphery of the B subunits (8Wilkens S. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 31804-31810Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 9Wilkens S. Inoue T. Forgac M. J. Biol. Chem. 2004; 279: 41942-41949Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 10Zhang Z. Inoue T. Forgac M. Wilkens S. FEBS Lett. 2006; 580: 2006-2010Crossref PubMed Scopus (35) Google Scholar). There is evidence that these elongated proteins densities are formed by the E and G subunits. First, it has been shown that these two subunits are able to form an elongated, heterodimeric complex with equimolar stoichiometry (11Fethiere J. Venzke D. Diepholz M. Seybert A. Geerlof A. Gentzel M. Wilm M. Böttcher B. J. Biol. Chem. 2004; 279: 40670-40676Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and second, chemical cross-linking from cysteines on the surface of the B subunits indicates close proximity to both E and G subunits from residues distributed between the bottom of the V1 and the very top (12Arata Y. Baleja J.D. Forgac M. Biochemistry. 2002; 41: 11301-11307Crossref PubMed Scopus (77) Google Scholar, 13Arata Y. Baleja J.D. Forgac M. J. Biol. Chem. 2002; 277: 3357-3363Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Recently, Kane and co-workers (14Ohira M. Smardon A.M. Charsky C.M. Liu J. Tarsio M. Kane P.M. J. Biol. Chem. 2006; 281: 22752-22760Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) have shown that there are at least two E and two G subunits per V1-ATPase complex. However, based on electron microscopic images of the intact V-ATPase and the isolated V1-ATPase domain, we had speculated earlier that the number of peripheral stalks might be three as, especially in images of the intact V-ATPase, each of the three B subunits seemed to have an elongated protein density bound at its periphery (8Wilkens S. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 31804-31810Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 9Wilkens S. Inoue T. Forgac M. J. Biol. Chem. 2004; 279: 41942-41949Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 10Zhang Z. Inoue T. Forgac M. Wilkens S. FEBS Lett. 2006; 580: 2006-2010Crossref PubMed Scopus (35) Google Scholar). vacuolar ATPase proton-pumping vacuolar ATPase water soluble domain of the vacuolar proton-pumping ATPase membrane-bound domain of the proton-pumping vacuolar ATPase mass spectrometry matrix assisted laser desorption ionization time-of-flight column volumes maltose-binding protein. V-ATPase activity is regulated in vivo by a reversible dissociation and reassociation mechanism, first described for the enzymes from yeast and insect (15Kane P.M. J. Biol. Chem. 1995; 270: 17025-17032Abstract Full Text Full Text PDF PubMed Google Scholar, 16Sumner J.P. Dow J.A. Earley F.G. Klein U. Jäger D. Wieczorek H. J. Biol. Chem. 1995; 270: 5649-5653Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) but now also observed for the V-ATPase of animal cells (17Sautin Y.Y. Lu M. Gaugler A. Zhang L. Gluck S.L. Mol. Cell. Biol. 2005; 25: 575-589Crossref PubMed Scopus (187) Google Scholar). During dissociation, the interaction between soluble and membrane domains involving the peripheral stalks has to be broken, and as a result of that process, subunit C dissociates from the separated V1 and V0 (18Curtis K.K. Francis S.A. Oluwatosin Y. Kane P.M. J. Biol. Chem. 2002; 277: 8979-8988Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To be able to understand the structural mechanism of enzyme dissociation, knowledge of the number of peripheral stalks and the nature of their interaction with the other subunits of the stalk domain and the complex are essential. We therefore decided to determine the copy number of the E and G subunits in the yeast V1-ATPase by two different mass spectrometry approaches. First, native electrospray ionization time-of-flight mass spectrometry was used to obtain a measurement of the molecular mass of the intact V1-ATPase complex that was accurate enough so that the subunit stoichiometry could be deduced. Second, we used known amounts of isotope-labeled subunits as internal standards to directly determine the copy number of subunits E and G in yeast V1. Results from both approaches were in excellent agreement and indicated that yeast V1-ATPase complex contains three copies each of the E and G subunits. Based on this result and our earlier electron microscopy images, we propose a structural model of the complex in which three peripheral stalks, via interaction with C, H, and a subunits, connect the V1 and V0 domains in intact yeast vacuolar ATPase. Yeast V1-ATPase PurificationV1-ATPase was purified from Saccharomyces cerevisiae strain SF838-5Aα vma10Δ::kanMX expressing a FLAG-tagged VMA10 in plasmid pRS315 (CEN6, LEU2) as described previously (19Zhang Z. Charsky C. Kane P.M. Wilkens S. J. Biol. Chem. 2003; 278: 47299-47306Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Briefly, one colony of the FLAG-Vma10p yeast strain was transferred into 5 ml of liquid SD leucine-dropout medium. The inoculum volume was gradually increased to 250 ml and then transferred into 8 liters of YPD in a fermenter (Electrolab). Yeast was grown to an A600 of 4-5 and harvested via low speed centrifugation. The cell pellet was resuspended in TBS (50 mm Tris, 150 mm NaCl, pH 7.4) at 1:1 w/v and frozen overnight at -20 °C. Frozen cell pellet was thawed in room temperature water, and protease inhibitors (1 μg/ml leupeptin, 1 μg/ml pepstatin A, 5 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride) and 1 mm dithiothreitol were added. Cells were lysed with 10 passes through a M110-L Microfluidizer (Microfluidics Corp.) cell disruptor. An additional 1 mm phenylmethylsulfonyl fluoride was added after the final pass. Crude lysate was centrifuged for 1 h at 250,000 × g, 4 °C, and the supernatant was passed over a 5-ml anti-FLAG M2 column (Sigma). The column was washed with 30 column volumes (CV) of TBS and eluted in 3 CV of TBS containing 100 μg/ml FLAG peptide. V1-containing fractions were pooled and concentrated down to 1 ml and further purified by size exclusion chromatography on a Superdex 75 HR 16/50 gel filtration column attached to an AKTA fast protein liquid chromatography system (GE Healthcare). Gel filtration was performed in TBS at a flow rate of 0.8 ml/min. V1-containing fractions were pooled and frozen in aliquots in liquid N2 for storage. Cloning of Yeast V-ATPase Subunits E and GAn Escherichia coli E subunit expression construct was generated as a fusion with maltose-binding protein (MBP) by subcloning the wild-type VMA4 open reading frame (minus the N-terminal Met) from S. cerevisiae genomic DNA into a pMAL-c2e vector (New England Biolabs) with a PreScission protease cleavage site in place of the stock enterokinase site, using the following primers: pM4, forward, 5′-GACAAGGTACCGTCCTCCGCTATTACTGCTTTTGAC-3′, and pM4, reverse, 5′-GTGCCAAGCTTCAATCAAAGAACTTTCTTGTCTTG-3′. Forward and reverse primers contained KpnI and HindIII digest sites, respectively. The resulting MBP fusion construct, pM4, was confirmed by DNA sequencing. pM4 construct was transformed into Rosetta 2 E. coli cells (Novagen) and plated on LB agar containing ampicillin and chloramphenicol. An E. coli FLAG-tagged G subunit expression construct was generated by subcloning the FLAG-tagged VMA10 open reading frame from the previously described pRS315 construct (19Zhang Z. Charsky C. Kane P.M. Wilkens S. J. Biol. Chem. 2003; 278: 47299-47306Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) into the first multiple cloning site of a pET-Duet-1 vector (Novagen), using the following primers: pDuet1G, forward, 5′-GATATACCATGGACTACAAGGACGACGATGA-3′, and pDuet1G, reverse, 5′-CATTATGCGGCCGCTTACAAGGCATTGATATGGACTTCAG-3′. Forward and reverse primers contained NcoI and NotI digest sites, respectively. The resulting construct, pG, was confirmed by DNA sequencing. pG construct was transformed into Rosetta 2 (DE3) E. coli cells (Novagen) and plated on LB agar containing ampicillin and chloramphenicol. Protein Expression and PurificationSingle colonies of pM4-expressing cells were picked and grown overnight at 37 °C in a 25-ml inoculum of LB containing antibiotics. The entire 25 ml was used to inoculate 1 liter of M9 medium containing 1 g of [15N]ammonium chloride (Spectra Stable Isotopes). 1 liter of culture was grown at 37 °C to an A595 = 0.6, at which point the temperature was lowered to 16 °C. After 1 h, culture was induced with 1 mm isopropyl-1-thio-d-galactopyranoside overnight at 16 °C. Cells were harvested by low speed centrifugation, resuspended in Column Buffer (20 mm Tris, 200 mm NaCl, 1 mm EDTA, pH 7) up to a final volume of 25 ml, and frozen overnight at -20 °C. Frozen cell pellet was thawed in room temperature water and treated with 1 μg/ml lysozyme and 10 μg/ml DNase I for 30′ on ice with gentle, intermittent shaking. Cells were sonicated for three cycles of 30 s on, 30 s off at 50% power using a Virtis VirSonic 100 sonicator and then centrifuged at 15,000 × g to clarify lysate. Lysate was diluted 1:5 in Column Buffer and applied to a 20-ml amylose resin column (New England Biolabs) at ∼1 ml/min, washed with 30 CV of Column Buffer, and then eluted in 1.5 CV of Column Buffer containing 10 mm maltose. Fractions containing MBP fusion protein were dialyzed in DEAE binding buffer (20 mm Tris, pH 8) overnight at 4 °C. MBP fusion was then passed over a 5-ml DEAE column, washed with 10 CV of DEAE binding buffer, and then eluted with a linear gradient from 0 to 100 mm NaCl over 40 CV. Fractions containing clean fusion protein were pooled and concentrated to a volume of 1 ml using Vivaspin 20 concentrator columns with a 50-kDa molecular mass cut-off. Fusion protein was then digested using PreScission protease (GE Healthcare) and 5 mm dithiothreitol overnight at 4 °C. E subunit was separated from MBP by gel filtration (Superdex 75 HR 16/50) in TBS. E subunit-containing fractions were pooled, aliquoted, and stored in liquid N2. For purification of subunit G, single colonies of pG-expressing cells were picked and grown overnight at 37 °C in a 25-ml inoculum of LB containing antibiotics. The entire 25 ml was used to inoculate 1 liter of M9 medium containing 1 g of [15N]ammonium chloride (Spectra Stable Isotopes). 1 liter of culture was grown at 37 °C to an A595 = 0.6 and induced with 1 mm isopropyl-1-thio-d-galactopyranoside for 4 h at 37 °C. Cells lysate was prepared as above, diluted 1:5 in TBS, and applied to a 5-ml anti-FLAG M2 column at ∼1 ml/min. The column was washed with 30 CV of TBS and eluted in 3 CV of TBS containing 100 μg/ml FLAG peptide. G subunit-containing fractions were pooled, concentrated to 1 ml, and subjected to gel filtration as above. Purified G subunit-containing fractions were pooled, aliquoted, and stored in liquid N2. Protein Concentration DeterminationProtein concentrations were routinely estimated by measuring UV absorbance in 6 m guanidine-HCl (20Gill S.C. von Hippel P. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5073) Google Scholar). For more accurate concentration determination, three samples each of V1 and individual E and G subunits were subjected to quantitative amino acid analysis (University of Texas Medical Branch). Electrospray Ionization Mass SpectrometryV1-ATPase sample was subjected to buffer exchange to 100 mm ammonium acetate, pH 6.8, by using an Ultrafree-0.5 centrifugal filter device with a cut-off of 10,000 Da (Millipore, Bedford). The sample was sprayed from solution of 2 μl containing ∼0.5 μm (∼0.3 mg/ml). The macromolecular mass spectrometry measurements were performed in positive ion mode using first generation modified Q-TOF 1 and LCT instruments (Micromass, Manchester, UK) (21van den Heuvel R.H. van Duijn E. Mazon H. Synowsky S.A. Lorenzen K. Versluis C. Brouns S.J. Langridge D. van der Oost J. Hoyes J. Heck A.J. Anal. Chem. 2006; 78: 7473-7483Crossref PubMed Scopus (206) Google Scholar, 22Lorenzen K. Versluis C. van Duijn E. van den Heuvel R. H.H. Heck A. J.R. Int. J. Mass Spectrom. 2007; 268: 198-206Crossref Scopus (59) Google Scholar). To detect intact gas-phase ions from large protein complexes, it is generally required to cool the ions collisionally by increasing the pressure in the first vacuum stages of the mass spectrometer (23Tahallah N. Pinkse M. Maier C.S. Heck A.J. Rapid Commun. Mass Spectrom. 2001; 15: 596-601Crossref PubMed Scopus (188) Google Scholar, 24Chernushevich I.V. Thomson B.A. Anal. Chem. 2004; 76: 1754-1760Crossref PubMed Scopus (206) Google Scholar). The pressures were optimized to balance preservation of noncovalent interactions and promote efficient ion desolvation in the interface region of the instrument. In this way, we were able to attain sharp ion signals enabling confident accurate mass determination, and consequently, the stoichiometry of the V1-ATPase complexes from the mass spectra. Furthermore, nanoelectrospray voltages were optimized for generation of the macromolecular protein complexes; the needle voltage was 1,500 V, and the sample cone voltage was 150 V. Mass Spectrometry of Individual Subunits of the V1-ATPaseV1-ATPase was denatured by diluting in 50% acetonitrile and 0.1% formic acid at a concentration of ∼0.5 μm. Mass measurements were performed in positive ion mode using an electrospray ionization time-of-flight instrument LCT (Micromass, Manchester, UK) essentially as described previously (22Lorenzen K. Versluis C. van Duijn E. van den Heuvel R. H.H. Heck A. J.R. Int. J. Mass Spectrom. 2007; 268: 198-206Crossref Scopus (59) Google Scholar). Isotope-labeled Subunit Titration and MALDI-Mass SpectrometryIncreasing amounts of 15N-labeled E and G subunits were mixed with a determined amount of yeast V1-ATPase, and the resulting protein mixture was separated on 12% SDS-PAGE gels. Bands containing both unlabeled and 15N-labeled E and G subunits were excised from the gels with a clean razor blade. Gel bands were diced into cubes of <1 mm3, washed three times in 400 μl of 25 mm ammonium bicarbonate, pH 8, 50% acetonitrile, shrunk in 100 μl of 100% acetonitrile, and dried for 30 min in a SpeedVac with heating. 25 μl of trypsin was added to the dried gel slices, and 25 mm ammonium bicarbonate was added to cover the swelled gel slices. After incubation overnight at 37 °C, the supernatant was transferred to a fresh Eppendorf tube, and the remaining gel slices were extracted twice with 50 μl of 50% acetonitrile, 5% trifluoroacetic acid in H2O for 15 min at room temperature. The combined extracts were dried for 1 h in a SpeedVac with heating. Dried peptide pellets were dissolved in 50% acetonitrile, 0.1% trifluoroacetic acid in H2O, purified with a ZipTip, and then pipetted onto a stainless steel MALDI probe at 1:4 and 1:10 dilutions in a saturated solution of α-cyano-4-hydroxycinnamic acid (Fluka) in 50% acetonitrile, 0.1% trifluoroacetic acid. Peptide samples were analyzed using a first generation Bruker Autoflex MALDI-TOF. Raw mass spectra were exported to CSV files using open source mMass software and processed using in-house scripts written in Ruby. Baseline was calculated as described previously (25Gras R. Müller M. Gasteiger E. Gay S. Binz P.A. Bienvenut W. Hoogland C. Sanchez J.C. Bairoch A. Hochstrasser D.F. Appel R.D. Electrophoresis. 1999; 20: 3535-3550Crossref PubMed Scopus (124) Google Scholar) with some modifications. Raw spectra were divided into 40-Da windows (si), within which both median (simed) and fifth percentile (silow) signal intensity values were calculated. Noise was defined as ni = 2(simed - silow) for a given window si. The local noise envelope for a given window si was defined as simed ± ni. The signal trend and noise envelope were calculated by cubic spline interpolation (26Gough B. 2nd Ed. GNU Scientific Library Reference Manual. Network Theory Ltd., Bristol, UK2006Google Scholar) of the si data points. The noise floor, or simed - ni, was subtracted from the intensity value of each data point to provide a baseline correction. The noise ceiling, or simed + ni, was used as a threshold to accept or reject peaks. The width of each isotopic envelope was defined as the maximum width that completely contained all peaks above the noise threshold. An isotopic envelope was rejected if the envelope was perturbed by noise or overlapping signal from other peptides. Mass spectra were obtained from digests of three gels each for E + V1 and G + V1 at various ratios. 4-8 peak pair ratios from validated pairs of isotopic envelopes were averaged together for each titration point for the G and E subunit, respectively. A 1:1 peak ratio of 14N- and 15N-containing peptides was interpolated using the least squares linear regression analysis. V1-ATPase Subunit Stoichiometry by Native Mass SpectrometryThe gentle nature of electrospray ionization and the spectacular advances in mass spectrometry instrumentation enable the direct analysis of large intact macromolecular protein complexes. This field, nowadays termed macromolecular or native mass spectrometry, focuses on the structural and functional analysis of the dynamics and interactions occurring in protein complexes. For this method, the sample of interest is electrosprayed from an aqueous solution of a volatile buffer such as ammonium acetate. Desolvation of the protein assemblies in the ion source interface generates multiply charged ions of the intact complexes that can be analyzed by the mass spectrometer. Native mass spectrometry has been used to obtain accurate information about stoichiometry, stability, and dynamics of protein complexes (21van den Heuvel R.H. van Duijn E. Mazon H. Synowsky S.A. Lorenzen K. Versluis C. Brouns S.J. Langridge D. van der Oost J. Hoyes J. Heck A.J. Anal. Chem. 2006; 78: 7473-7483Crossref PubMed Scopus (206) Google Scholar, 27Loo J.A. Mass Spectrom. Rev. 1997; 16: 1-23Crossref PubMed Scopus (1165) Google Scholar, 28Robinson C.V. Nat. Struct. Biol. 2002; 9: 505-506Crossref PubMed Scopus (17) Google Scholar, 29Heck A.J. Van den Heuvel R.H. Mass Spectrom. Rev. 2004; 23: 368-389Crossref PubMed Scopus (507) Google Scholar, 30van den Heuvel R.H. Heck A.J. Curr. Opin. Chem. Biol. 2004; 8: 519-526Crossref PubMed Scopus (259) Google Scholar, 31Hernandez H. Dziembowski A. Taverner T Seraphin B Robinson C.V. EMBO Rep. 2006; 7: 605-610Crossref PubMed Scopus (159) Google Scholar, 32Synowsky S.A. van den Heuvel R.H. Mohammed S. Pijnappel P.W. Heck A.J. Mol. Cell. Proteomics. 2006; 5: 1581-1592Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Here, we applied macromolecular mass spectrometry to investigate the composition of V1-ATPase from the yeast S. cerevisiae. Before the analysis of V1-ATPase under pseudophysiological native solvent conditions, we first analyzed the complex under denaturing solvent conditions. From the resulting mass spectra, we were able to determine the accurate masses of each subunit present in the complex. The obtained subunit masses are given in Table 1, together with the predicted masses of the subunits derived from the gene sequences. The only subunit not detected by this approach was subunit D. The absence of subunit D in the mass spectra might be due to the relatively hydrophobic nature of the polypeptide. On the basis of the gene-predicted amino acid sequences, we concluded that most subunits lacked the N-terminal methionine residue, except subunit G, which in our purifications also contains the N-terminal FLAG tag. For subunits B and H, the masses were very close to the expected masses. For the others, we observed slightly higher experimental masses, with mass increases likely to be related to post-translational modifications, such as N-terminal acetylation (+42 Da).TABLE 1Comparison from theoretical and measured masses of V1-ATPase-associated proteins and the intact- and subunit depleted V1-ATPase complexesExperimental massesTheoretical massesSubunitsMass differencesDaaStandard errors represent the maximum mass error obtained from the full width at half-maximum of the most intense peak for each charge state distributionDaDa67,642.1 ± 28.067,592.5A+49.657,630.5 ± 32.257,618.2B+12.3–29,063.0D–26,387.3 ± 4.526,340.2E+47.113,374.1 ± 1.013,330.2F+43.913,752.4 ± 1.313,707.6GbN-terminal Met + FLAG tag+44.854,298.1 ± 22.154,284.8H+13.3ComplexesNameComposition593,576 ± 3,000592,454IA3B3DE3FG3H+1,122553,544 ± 2,000552,406IIA3B3DE2FG2H+1,138428,305 ± 1,600427,195IIIA2B2DE2FG2H+1,110a Standard errors represent the maximum mass error obtained from the full width at half-maximum of the most intense peak for each charge state distributionb N-terminal Met + FLAG tag Open table in a new tab Next, we investigated V1-ATPase by macromolecular mass spectrometry. Fig. 1 shows a representative native mass spectrum of yeast V1-ATPase obtained from an aqueous solution of the protein in 100 mm ammonium acetate, pH 6.8. The spectrum reveals three individual charge state distributions centered around m/z values of 10,000, 11,400, and 11,500, respectively, with the most abundant distribution around m/z 11,500. The protein mass could be easily determined by using the well resolved multiple charge states of the protein. Thus, mass determination of the ion series with the highest m/z values (around 11,500) yielded a molecular mass of 593,576 ± 3,000 Da (Table 1, complex I). When we sum the theoretical masses of each subunit (i.e. as predicted from the gene sequences) in the stoichiometry A3B3DE3FG3H, we obtain a mass of 592,454 Da, which is a very close match to the observed mass of the complex. This mass is only 0.19% higher than the measured mass for this complex. The observed deviation between the theoretical and experimental mass can be partly explained by the fact that we observed higher experimental masses for each individual subunit under denaturing conditions but also by incomplete desolvation, which may leave several water or buffer molecules attached to the protein complex (29Heck A.J. Van den Heuvel R.H. Mass Spectrom. Rev. 2004; 23: 368-389Crossref PubMed Scopus (507) Google Scholar, 33Sobott F. McCammon M.G. Hernandez H. Robinson C.V. Philos. Transact. A Math. Phys. Eng. Sci. 2005; 363: 379-389PubMed Google Scholar). To address the first issue, if we sum the experimental derived masses of the subunits in an A3B3DE3FG3H stoichiometry, we come to a mass of 592,971 Da, a value that is now only off by 0.1%. Therefore, we conclude with high confidence that the subunits A, B, E, and G are present in three copies in this complex I, whereas D, F, and H are only present as a single copy. The second ions series centered around m/z 11,400 had a determined mass of 553,544 ± 2,000 Da (Table 1, complex II). The closest theoretical matching mass is 552,406 Da, corresponding to a complex of A3B3DE2FG2H stoichiometry. Thus, when compared with the most abundant complex I, the complex II lacks one copy of E and one copy of G. The last ion series centered around m/z values of 10,000 had a mass of 428,305 ± 1,600 Da (Table 1, complex III). Here, we can unambiguously assign the mass to a subcomplex of V1-ATPase with a stoichiometry of A2B2DE2FG2H (427,195 Da). Thus, in contrast to complex I, the subunits A, B, E, and G are present only in two copies in the complex III. The zero charge convoluted mass spectrum (Fig. 1, inset) semiquantifies the relative abundance of each complex in our purification. We estimated that in the preparation used for electrospray mass spectrometry, the complexes I, II, and III represented 60, 15, and 25%, respectively. In two other experiments, the estimated ratios were 57/17/26% and 72/12/16% for complexes I, II, and II, respectively (not shown). This indicates some variability in the integrity of the V1-ATPase, possibly due to sample preparation and/or electrospray ionization (see below). Nevertheless, the data show that the majority of V1-ATPase complexes have three copies of E and three copies of G. We also hypothesize from our data that the subunits E and G are strongly correlated, consistent with these two subunits forming a heterodimer because in the complexes I, II, and III, each time the copy numbers for E and G were equal. Only very minor amounts of individual E and G subunits could be seen in the low m/z range in the native mass spectra of yeast V1-ATPase, suggesting that the two subcomplexes II and III were already p
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