Artigo Acesso aberto Revisado por pares

Tandem Phosphorylation of Ser-911 and Thr-912 at the C Terminus of Yeast Plasma Membrane H+-ATPase Leads to Glucose-dependent Activation

2007; Elsevier BV; Volume: 282; Issue: 49 Linguagem: Inglês

10.1074/jbc.m706094200

ISSN

1083-351X

Autores

Silvia Lecchi, Clark J. Nelson, Kenneth E. Allen, Danielle L. Swaney, Katie L. Thompson, Joshua J. Coon, Michael R. Sussman, Carolyn W. Slayman,

Tópico(s)

Mitochondrial Function and Pathology

Resumo

In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H+-ATPase, which is responsible for H+-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5–10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896–918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal 14N/15N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H+-ATPase. In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H+-ATPase, which is responsible for H+-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5–10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896–918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal 14N/15N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H+-ATPase. Pma1 H+-ATPase of Saccharomyces cerevisiae belongs to the widely distributed family of P2-type cation pumps, which include the sarcoplasmic reticulum and plasma membrane Ca2+-ATPases and the Na+,K+-ATPase of animal cells and the plasma membrane H+-ATPase of higher plants (1Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (418) Google Scholar). Like other members of the P2 family, the 100-kDa yeast ATPase is folded into 3 cytoplasmic domains (N, P, and A) that form the catalytic portion of the molecule, anchored in the lipid bilayer by 10 transmembrane helices that comprise the ion transport pathway (2Kuhlbrandt W. Zeelen J. Dietrich J. Science. 2002; 297: 1692-1696Crossref PubMed Scopus (101) Google Scholar). Pma1 ATPase acts physiologically to pump protons out of the cell, creating the electrochemical gradient that drives solute uptake by an array of H+-coupled cotransporters. Its activity is strongly regulated, reflecting both its essential role in cell growth and the fact that, as the most abundant protein in the plasma membrane, it consumes at least 20% of cellular ATP (3Morsomme P. Slayman C.W. Goffeau A. Biochim. Biophys. Acta. 2000; 1469: 133-157Crossref PubMed Scopus (103) Google Scholar). The first evidence for Pma1 regulation was reported in 1983 by Serrano (4Serrano R. FEBS Lett. 1983; 156: 11-14Crossref PubMed Scopus (305) Google Scholar), who found that the addition of glucose to carbon-starved yeast cells led within minutes to a 5–10-fold increase in ATPase activity accompanied by a severalfold lowering of the Km for MgATP and an alkaline shift in pH optimum. Given the rapidity of the activation, it seemed likely to occur at the post-translational level. Direct support for this idea came in 1991, when Chang and Slayman (5Chang A. Slayman C.W. J. Cell Biol. 1991; 115: 289-295Crossref PubMed Scopus (151) Google Scholar) immunoprecipitated Pma1 ATPase from 32P-labeled yeast cells and analyzed it by two-dimensional phosphopeptide mapping. Against a background of "constitutive" phosphorylation, which occurred in a stepwise fashion as newly synthesized ATPase moved from the endoplasmic reticulum through the Golgi and secretory vesicles to the cell surface, additional phosphorylation took place when glucose was added to carbon-starved cells. Complete digestion of the 32P-labeled ATPase revealed both phosphoserine and phosphothreonine but no detectable phosphotyrosine (5Chang A. Slayman C.W. J. Cell Biol. 1991; 115: 289-295Crossref PubMed Scopus (151) Google Scholar). These results strongly supported the idea that glucose activation of the ATPase was related to kinase-mediated phosphorylation at one or more Ser/Thr residues, although the location of the phosphorylation sites had yet to be determined. In parallel, studies in several laboratories sought to identify the domain(s) of the 100-kDa Pma1 ATPase that participates in glucose-dependent regulation (for review, see Ref. 6Portillo F. Biochim. Biophys. Acta. 2000; 1469: 31-42Crossref PubMed Scopus (163) Google Scholar). Attention soon focused on the 45-amino acid C-terminal tail, which is known to protrude from the plasma membrane into the cytoplasm (7Mandala S.M. Slayman C.W. J. Biol. Chem. 1989; 264: 16276-16281Abstract Full Text PDF PubMed Google Scholar). Removal of the last 18 amino acids by mutagenesis led to constitutive activation of the ATPase even in the absence of glucose (8Mason A.B. Kardos T.B. Monk B.C. Biochim. Biophys. Acta. 1998; 1372: 261-271Crossref PubMed Scopus (15) Google Scholar, 9Portillo F. de Larrinoa I.F. Serrano R. FEBS Lett. 1989; 247: 381-385Crossref PubMed Scopus (99) Google Scholar). Consistent with this observation, immunoblotting with anti-C-terminal antibody revealed that the C terminus was relatively inaccessible during carbon starvation but could be rapidly digested by trypsin upon the addition of glucose (10Lecchi S. Allen K.E. Pardo J.P. Mason A.B. Slayman C.W. Biochemistry. 2005; 44: 16624-16632Crossref PubMed Scopus (44) Google Scholar). Of added significance was the fact that amino acid substitutions within the tail, especially at Thr-912, reduced glucose activation in a manner that could be suppressed by second-site mutations elsewhere in the 100-kDa protein (11Portillo F. Eraso P. Serrano R. FEBS Lett. 1991; 287: 71-74Crossref PubMed Scopus (88) Google Scholar, 12Eraso P. Portillo F. J. Biol. Chem. 1994; 269: 10393-10399Abstract Full Text PDF PubMed Google Scholar). Replacing Thr-912 with Ala (to prevent phosphorylation) arrested the ATPase in a trypsin-resistant state characteristic of carbon-starved wild-type cells, whereas replacing it with Asp (to mimic phosphorylation) locked the ATPase in a trypsin-sensitive state typical of glucose-activated wild-type cells (10Lecchi S. Allen K.E. Pardo J.P. Mason A.B. Slayman C.W. Biochemistry. 2005; 44: 16624-16632Crossref PubMed Scopus (44) Google Scholar). Taken together, these results pointed to a key regulatory role of the C-terminal tail, which appears to act in an autoinhibitory fashion during glucose starvation, binding tightly to regions of the N and/or P domains to inhibit ATPase activity. Upon the addition of glucose, the C-terminal tail is released from autoinhibition, most likely through phosphorylation, and allows the ATPase to assume an open, activated conformation. In the work described below, we have used an ensemble of state-of-the-art mass spectrometric methods to map phosphorylation sites throughout the 100-kDa Pma1 ATPase, with the aim of directly identifying the site(s) responsible for glucose activation. The results point conclusively to Ser-911 and Thr-912 in the C-terminal tail, acting in concert as phosphorylation sites that mediate the glucose effect. Yeast Strains—Strain NY13 of S. cerevisiae (MATa ura3-52) was used in this study. For metabolic labeling (see below) the wild-type URA3 allele was integrated downstream from the PMA1 locus to confer growth in the absence of added uracil. Preparation of Plasma Membranes from Carbon-starved and Glucose-metabolizing Cells—Cells were grown to mid-exponential phase at 30 °C in minimal medium containing 4% glucose, harvested, washed twice with water, and incubated with shaking for 1 h in water without glucose. Glucose (4%) was then added back to an aliquot of the culture for 30 min (glucose-metabolizing cells (GM)), 2The abbreviations used are: GM, glucose-metabolizing cells; CS, carbon-starved cells; MALDI TOF, matrix-assisted laser desorption ionization time-of-flight; PLB, phospholamban; SERCA, sarcoplasmic reticulum Ca2+-ATPase; MS, mass spectroscopy; HPLC, high performance liquid chromatography; QTOF, quadrupole-time of flight; ETD, electron transfer dissociation. whereas another aliquot was kept in glucose-free medium (carbon-starved cells (CS)). Plasma membranes were prepared from both aliquots as described by Perlin et al. (13Perlin D.S. Harris S.L. Seto-Young D. Haber J.E. J. Biol. Chem. 1989; 264: 21857-21864Abstract Full Text PDF PubMed Google Scholar) with the addition of phosphatase inhibitors (1 mm NaF and 2 mm ammonium molybdate) to each buffer at every step of the preparation. Plasma membranes were resuspended in 1 mm EGTA/Tris and 20% glycerol (pH 7.5) containing phosphatase inhibitors (see above) and protease inhibitor mixture (pepstatin, aprotin, leupeptin, and chymostatin, each at 10 μg/ml). All procedures were carried out at 4 °C. Protein concentrations were determined by the method of Lowry et al. (14Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Phosphatase Treatment—Where indicated, aliquots of 50 μg of plasma membranes were treated with 50 units of calf intestinal alkaline phosphatase (New England Biolabs) at 37 °C for 14 h in the presence of protease inhibitors. Sample Preparation for MS Analysis—Plasma membranes (100 μg) from CS and GM cells were individually subjected to SDS-PAGE and stained with Coomassie Blue, and the 100-kDa Pma1 band was excised, cut into 1-mm3 pieces, and placed in a siliconized tube. The gel pieces were destained with brief washes in 100 mm NH4HCO3, 50% methanol, then dehydrated for 5 min in 25 mm NH4HCO3, 50% acetonitrile and for 30 s in 100% acetonitrile and dried in a vacuum centrifuge for 5 min. Samples were rehydrated in a solution containing 25 mm dithiothreitol in 25 mm NH4HCO3 and reduced for 20 min at 56 °C. Protein alkylation was performed by adding freshly prepared iodoacetamide in 25 mm NH4HCO3 and incubating for 20 min at room temperature in the dark. Gel pieces were dehydrated and dried again as described above. To perform in-gel digestion, the gel pieces were rehydrated in 25 mm NH4HCO3, 3% acetonitrile containing 20 ng/μlof trypsin (Promega sequencing grade modified), Lys-C (Roche Applied Science sequencing grade), chymotrypsin (Worthington), Glu-C, or Asp-N (Sigma) and incubated overnight at 37 °C. Digested peptides were extracted by vortexing for 15 min in 0.1% trifluoroacetic acid. A second extraction was performed using 70% acetonitrile, 25% H2O, 5% trifluoroacetic acid. Extracted peptides from survey samples were dried in a vacuum centrifuge and reconstituted in 0.1% trifluoroacetic acid (15Jimenez, C. R., Huang, L., Qiu, Y., and Burlingame, A. L. (1998) in Current Protocols in Protein Science (Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T., eds) pp. 4.1–4.5, John Wiley & Sons, New YorkGoogle Scholar). The samples were then solid phase-extracted using C18-Zip-Tips (Millipore). Metabolic Labeling with a Stable Isotope—Non-labeled cells (for the 14N samples) were grown in minimal medium prepared with regular yeast nitrogen base lacking amino acids. For the 15N-labeled samples, cells were grown in minimal medium prepared with yeast nitrogen base lacking amino acids and ammonium sulfate to which [15N]ammonium sulfate (99 atom %; Isotec) had been added. The rest of the experiment was carried out as described under "Results." MALDI TOF/TOF Mass Spectrometry—A Sciex 4800 MALDI TOF/TOF (Applied Biosystems) was used to analyze the in-gel digests. An MS scan was conducted from 700 to 4000 m/z, and the 10 most abundant peaks with signal to noise ratios >10 were selected for tandem mass spectrometry (MS/MS) experiments. Data were analyzed using Explorer 4000 software (Version 3.6) and searched with Mascot 2.1 (Matrix Science) against a list of yeast protein sequences downloaded from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) (16Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6814) Google Scholar). Searches allowed up to two missed cleavages for a given enzyme and MS and MS/MS tolerances of ±0.4 Da. Variable modifications included carbamimidomethylation of cysteine, deamidation of asparagine and glutamine, oxidation of methionine, phosphorylation of serine, threonine, and tyrosine, and N-terminal acetylation of the protein. Three-dimensional Ion-trap Mass Spectrometry—In-gel-digested samples were analyzed with an Agilent 1100 series LC/MSD ion trap mass spectrometer. Samples were loaded with an autosampler (Agilent) onto a C18 reverse-phase trap cartridge (Agilent). After loading, the trap cartridge was switched in-line with an analytical 75-μm × 150-mm column packed with 3.5-μm Zorbax C18 reversed-phase particles (Agilent) and a gradient conducted with an Agilent 1100 series capillary HPLC system. Peptides were eluted with a gradient of buffer A (0.1% v/v formic acid) and buffer B (95% v/v acetonitrile and 0.1% v/v formic acid) by 5–60% buffer B in 90 min. The instrument was operated in a data-dependent fashion with an MS survey scan (300–2200 m/z) followed by MS/MS or MS/MS/MS analysis (300–2200 m/z) of as many as the five most intense peaks with dynamic exclusion for 120 s of fragmented m/z values. All ion trap MS2 and MS3 data were converted to Mascot generic files with DataAnalysis 2.2 (Agilent) using default settings. Mascot generic format (MGF) files were then searched using Mascot with the same parameters as described above except that MS and MS/MS tolerances were set to ±0.8 Da. QTOF Mass Spectrometry—Trypsin, Lys-C, chymotrypsin, and Glu-C digests were analyzed using a QTOF2 mass spectrometer (Micromass) coupled to an HP 1100 HPLC (Agilent). Separations were conducted using home-pulled fused silica columns (100 μm × 10 cm) packed with Eclipse C18 particles (Agilent) and eluted at ∼500 nl/min with a gradient of buffer A (0.1% formic acid v/v) and buffer B (95% v/v acetonitrile, 0.1% v/v formic acid). After loading samples in 5% buffer B, the gradient was 5–12% buffer B in 10 min, 12–50% buffer B in 105 min, 50–60% buffer B in 5 min, and 60–100% buffer B in 5 min. The instrument was operated in a data-dependent fashion with an MS scan (400–2200 m/z) with as many as 2 m/z values selected for MS/MS experiments (50–2200 m/z). After being selected for tandem MS, individual m/z peaks within 2 Da were dynamically excluded for 120 s. QTOF MS/MS data were converted to peak list files using ProteinLynx Global Server 2.15 (Waters). The MS and MS/MS scans were smoothed twice with a Savitzky-Golay smooth. MS scans were background-subtracted using the standard method with a first-order polynomial, whereas MS/MS data were background-subtracted with the adaptive method and a fifth-order polynomial, and the data were saved as peak lists. Peak list files were then searched with Mascot using the same parameters as described above except that MS and MS/MS tolerances were set to ±0.3 Da. Isotopic Measurements—Metabolizing cells were grown in standard (14N) medium and starving cells in 15N-enriched medium (two experiments), and a third experiment was performed but with the labels reversed. Combined samples were analyzed by QTOF mass spectrometry (see above), and data were processed as previously described (17Nelson C.J. Huttlin E.L. Hegeman A.D. Harms A.C. Sussman M.R. Proteomics. 2007; 7: 1279-1292Crossref PubMed Scopus (93) Google Scholar). Briefly, samples were searched using Mascot 2.0 with amino acid masses corresponding to natural abundance (14N) and then searched again with amino acid masses corresponding to 15N-labeling. Both sets of searches were conducted with the same modifications as those used for isotopically unlabeled samples except that deamidation was not allowed in the 15N searches because this modification would be indistinguishable due to the isotopic label. The results from heavy and light searches were combined, and extracted ion chromatograms were generated 0.25 m/z wide for the monoisotopic m/z value and the 100% 15N incorporation of identified peptides. In addition, extracted ion chromatograms were generated for m/z values corresponding to the double-, triple-, quadruple, and quintuple-charged forms of the 896STRSVEDFMAAMQRVSTQHET918 peptide with zero, one, and two phosphate moieties. Linear regression was used to calculate a 15N/14N ratio using extracted ion chromatogram values for the monoisotopic peak and the peak corresponding to the peptide with 100% 15N incorporation using Mathematica 5.1 (Wolfram Research). Ratios with an R-value greater than 0.85 were used to determine GM:CS measurements. For a given phosphorylation state of the peptide, the measurements from all charge states were averaged. Because the monoisotopic peak and the 100% 15N incorporation peak represent different fractions of their respective isotopic envelopes, a correction was calculated and applied to the measured ratio. Localization of Phosphorylation Sites by Electron Transfer Dissociation (ETD)-MS/MS—An ETD-enabled ThermoFisher linear ion trap-orbitrap hybrid mass spectrometer was employed to determine the exact sites of phosphorylation on peptides P1 and P2 from CS and GM samples (18McAlister G.C. Phanstiel D. Good D.M. Berggren W.T. Coon J.J. Anal. Chem. 2007; 79: 3525-3534Crossref PubMed Scopus (145) Google Scholar). Peptide separations were performed on a reversed-phase, self-prepared capillary column. The separation column consisted of a precolumn that was butt-connected to an analytical column using a 0.012-inch inner diameter Teflon sleeve (Zeus Industrial Products, Orangeburg, SC). The analytical column was 360 μm × 50 μm (outer diameter × inner diameter) fused silica (Polymicro Technologies, Phoenix, AR) and was prepared by pulling a bottle neck and integrated ESI tip using a laser puller (Sutter Instrument Co., Novarto, CA, model P-2000) as described elsewhere (19Martin S.E. Shabanowitz J. Hunt D.F. Marto J.A. Anal. Chem. 2000; 72: 4266-4274Crossref PubMed Scopus (303) Google Scholar). Approximately 1–2 mm of 5–20-μm C18 particles (YMC, Milford, MA) followed by 7 cm of 5-μm C18 particles (Alltech Associates Inc., Deerfield, IL) were packed into the analytical column. The precolumn was made of 360-μm × 75-μm (outer diameter × inner diameter) fused silica and incorporated a LiChrosorb frit (EMD Chemicals Inc., Gibbstown, NJ) of ∼2 mm in length. The precolumn was then packed with 5 cm of 5-μm C18 particles. Approximately 2 pmol of Lys-C-digested Pma1 were loaded onto the column, and on-line peptide separations were performed with an Agilent 1100 Series binary HPLC system coupled to the ETD-enabled orbitrap. The sample was eluted from the column at a flow rate of 60 nl/min into the mass spectrometer using a linear gradient of buffer A (0.1 m acetic acid) and buffer B (70% v/v acetonitrile in 0.1 m acetic acid) at 0–100% B in 60 min. The mass spectrometer was operated in a targeted ion fashion. First, a full scan (300–2000 m/z) mass spectrum was acquired using the orbitrap at a nominal resolving power of 30,000. The following precursor cations were isolated and subjected to a 100-ms ETD reaction: 668.1, 676.6, 688.1, 696.6, 708.1, 716.6 m/z. These ions corresponded to the unphosphorylated, singly phosphorylated, and doubly phosphorylated forms of the 896–918 peptide that were 14N- and 15N-labeled in the [M + 4H]4+ charged state. Relevant scans were manually sequenced. Recovery of Peptide Fragments for Analysis—As described in the Introduction, the central goal of this study was to identify sites of protein phosphorylation that are mechanistically related to the activation of Pma1 ATPase by glucose. The first step was to prepare plasma membranes from CS and GM cells, purify the ATPase, subject it to in-gel proteolytic digestion, and perform mass spectrometric analyses. To maximize coverage of the protein, five different proteases were used: trypsin, Lys-C, chymotrypsin, Glu-C, and Asp-N. Fig. 1 is a topological map of the ATPase showing that peptides corresponding to 78% of the cytoplasmic part of the protein were identified (see the supplemental Appendix for a complete table of results). Most of the undetected peptides were located in the 10 transmembrane segments of the ATPase, where the predicted proteolytic fragments are generally too large and/or too hydrophobic to be observed by MS analysis. Two regions of the N-terminal extension (Asp-31—Gly-70 and Asp-83—Lys-98) and the short loop between transmembrane segments 6 and 7 were also missing from the list of recovered peptides. Differential Phosphorylation in the C-terminal Tail Correlates with Glucose Activation of Pma1—As illustrated in Fig. 2, peptide 896–918 (P0; m/z 890.5, 2671.5 Da), obtained after digestion with Lys-C, was identified by LC-MS/MS using the three-dimensional ion trap mass spectrometer in both CS and GM samples. This peptide corresponds to the C-terminal tail of the ATPase: 896STRSVEDFMAAMQRVSTQHEKET918. Both CS and GM samples also contained peptide 896–916 (2437.15 Da), in which Lys-C cleavage had occurred at Lys-916. The signal for 896–916 was much less intense than that for 896–918, presumably reflecting the fact that cleavage at Lys-916 was strongly inhibited by the flanking Glu residues (20Thiede B. Lamer S. Mattow J. Siejak F. Dimmler C. Rudel T. Jungblut P.R. Rapid Commun. Mass Spectrom. 2000; 14: 496-502Crossref PubMed Scopus (118) Google Scholar). Furthermore, peptide 896–916 is found in Pma2, a poorly expressed isoform of yeast plasma membrane H+-ATPase (21Schlesser A. Ulaszewski S. Ghislain M. Goffeau A. J. Biol. Chem. 1988; 263: 19480-19487Abstract Full Text PDF PubMed Google Scholar), whereas peptide 896–918 is unique to Pma1. For both reasons, most of the analysis focused on 896–918. In addition to P0, both the carbon-starved and glucose-metabolizing samples contained a peptide whose mass was ∼80 Da heavier than the unmodified 896–918, as expected for a singly phosphorylated form (P1; m/z 917.2). The glucose-metabolizing sample also contained a peptide whose mass was consistent with a doubly phosphorylated form of 896–918 (P2; m/z 943.8). Neither sample gave any detectable evidence for higher multiples of phosphorylation of peptide 896–918. When peptides P1 and P2 were analyzed by MS/MS on the three-dimensional ion trap, the spectrum for P1 displayed a single dominant peak corresponding to a neutral loss of 98 Da from the parent peptide, whereas the spectrum for P2 showed two abundant peaks, corresponding to the loss of one and two phosphate moieties (Fig. 3). This is the expected signature for phosphopeptides. Unlike most peptides that undergo random protonation along the backbone in the gas phase to generate a series of sequence-informative fragments by collisional activation, phosphopeptides are preferentially cleaved at the phosphate group, leaving the peptide backbone intact (22Coon J.J. Ueberheide B. Syka J.E. Dryhurst D.D. Ausio J. Shabanowitz J. Hunt D.F. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9463-9468Crossref PubMed Scopus (341) Google Scholar). Taken together the results are consistent with the idea that the C-terminal tail of Pma1 ATPase is singly phosphorylated in carbon-starved cells and undergoes a second phosphorylation during glucose activation. However, the lack of peptide backbone fragmentation upon collisional activation made it impossible to locate the sites of phosphorylation in the P1 and P2 peptides by this approach. Locating the Phosphorylation Sites—We, therefore, turned to a new peptide fragmentation method known as ETD (18McAlister G.C. Phanstiel D. Good D.M. Berggren W.T. Coon J.J. Anal. Chem. 2007; 79: 3525-3534Crossref PubMed Scopus (145) Google Scholar, 22Coon J.J. Ueberheide B. Syka J.E. Dryhurst D.D. Ausio J. Shabanowitz J. Hunt D.F. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9463-9468Crossref PubMed Scopus (341) Google Scholar, 23Coon J.J. Syka J.E.P. Schwartz J.C. Shabanowitz J. Hunt D.F. Int. J. Mass Spectrom. 2004; 236: 33-42Crossref Scopus (182) Google Scholar, 24Coon J.J. Syka J.E. Shabanowitz J. Hunt D.F. Biotechniques. 2005; 38 (521, and 523): 519Crossref PubMed Scopus (87) Google Scholar, 25Syka J.E. Coon J.J. Schroeder M.J. Shabanowitz J. Hunt D.F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9528-9533Crossref PubMed Scopus (2014) Google Scholar) to identify the two phosphorylation sites. ETD is performed in an ion-trap mass spectrometer by reacting isolated peptide cation precursors with small-molecule radical anions. The reaction results in the transfer of an electron to the peptide cation, which then dissociates randomly to generate a series of peptide backbone cleavages while preserving labile posttranslational modifications such as phosphorylation. The present study employed a new instrument equipped with a secondary orbitrap mass analyzer, allowing the intact peptide mass to be recorded with high m/z resolution and accuracy. Peptide P1 was sequenced from an 14N-labeled CS sample and an 15N-labeled GM sample that had been mixed together before the isolation of plasma membranes (see next section). Inspection of the ETD-MS/MS spectra revealed that 14N-labeled peptide P1 from CS cells was phosphorylated at Thr-912 (Fig. 4A). Identical results were seen for the 15N-labeled P1 from GM cells (Fig. 4B). By contrast, peptide P2, which was found only in samples from GM cells, was phosphorylated at both Ser-911 and Thr-912 (Fig. 5A–C). In parallel, the shorter peptide 896–916 from GM samples was also found to be phosphorylated at both Ser-911 and Thr-912 (Fig. 5D). Thus, Thr-912 appears to be a constitutive site of phosphorylation in Pma1 ATPase, whereas Ser-911 becomes phosphorylated during glucose activation.FIGURE 5ETD-MS/MS spectra of peptide P2 from the C-terminal tail of Pma1 ATPase, demonstrating phosphorylation on both Ser-911 and Thr-912. GM cells were grown for this experiment in 15N-enriched medium and doubly phosphorylated forms of peptide 896–918 (panels A–C) and peptide 896–916 (panel D) were studied. A, within the isolation window of the target peptide precursor cation, a co-eluting peptide cation could be seen with the same nominal m/z value but a different charge state (peptide 2, m/z value 716.349). The isotopic cluster m/z peaks corresponding to the doubly phosphorylated form of Pma1 peptide 896–918 are marked with open circles, whereas those corresponding to the co-eluting peptide are marked with closed circles. B, upon ETD fragmentation, c- and z-type product ions were generated from both peptide precursor cations. From these fragments, the target peptide and sites of phosphorylation (Ser-911 and Thr-912) could readily be determined. Fragment ions arising from the co-eluting peptide cation were also observed and have been denoted with an asterisk. C, calculation of the theoretical mass and isotopic distribution for the identified phosphopeptide revealed an identical match to the observed full MS spectrum and differed by only 5 ppm. Note that the y axis was magnified 50-fold in regions marked by brackets. D, ETD-MS/MS spectra of the doubly phosphorylated form of peptide 896–916. Analysis of consecutive backbone fragment ions from either the C terminus (z-type) or N terminus (c-type) made it possible to identify the phosphorylation site on this peptide as Ser-911 and Thr-912.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To make certain that Thr-912 was the only phosphorylation site in peptide P1, the c- and z-type ions were carefully examined along the peptide backbone. The c-type ion series did not exhibit the +80 addition until c7; likewise, the z-type ion series exhibited a +80 addition at z17. To rule out the possibility of a second isoform, singly phosphorylated at Ser-911, we searched for the presence of unmodified c7 and z17, but no m/z peaks corresponding to those values were detected. In addition to Ser-911 and Thr-912, the C-terminal tail of Pma1 AT

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