Identification of New Intrinsic Proteins in Arabidopsis Plasma Membrane Proteome
2004; Elsevier BV; Volume: 3; Issue: 7 Linguagem: Inglês
10.1074/mcp.m400001-mcp200
ISSN1535-9484
AutoresAnne Marmagne, Marie-Aude Rouet, Myriam Ferro, Norbert Rolland, Carine Alcon, Jacques Joyard, Jérôme Garin, Hélène Barbier‐Brygoo, Geneviève Ephritikhine,
Tópico(s)Ubiquitin and proteasome pathways
ResumoIdentification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies. Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies. The plasma membrane (PM) 1The abbreviations used are: PM, plasma membrane; Cyt b5, cytochrome b5; 2-D, two-dimensional; GFP, green fluorescent protein; GRAVY, grand average of hydropathy; pI, isoelectric point; TM, transmembrane segment; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; LC, liquid chromatography; PEG, polyethyleneglycol; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol; C/M, chloroform/methanol; TIP, tonoplast intrinsic protein; PIP, PM intrinsic proteins; VDAC, voltage-dependent anion channel. 1The abbreviations used are: PM, plasma membrane; Cyt b5, cytochrome b5; 2-D, two-dimensional; GFP, green fluorescent protein; GRAVY, grand average of hydropathy; pI, isoelectric point; TM, transmembrane segment; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; LC, liquid chromatography; PEG, polyethyleneglycol; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol; C/M, chloroform/methanol; TIP, tonoplast intrinsic protein; PIP, PM intrinsic proteins; VDAC, voltage-dependent anion channel. is an organized system serving as a structural and communication interface with the extracellular environment for exchanges of information and substances. In animal cells, PM proteins represent a point for potential therapeutic intervention, making the PM a source of drug targets, for instance in cancer research (1Harvey S. Zhang Y. Landry F. Miller C. Smith J.W. Insights into a plasma membrane signature..Physiol. Genomics. 2001; 5: 129-136Google Scholar). In plant cells too, as signaling processes controlling responses to biotic and abiotic factors occur in PM, a better knowledge of the PM proteome would help developing defense strategies. Indeed, in plant cells as well as in animal cells, the PM is controlling many primary cellular functions, such as metabolite and ion transport, endocytosis, cell differentiation and proliferation, etc. All these processes involve a large array of proteins with highly diverse structure and function. In addition, the strength of their association to the membrane varies, some being well embedded in the membrane lipid core while others are more peripheral proteins, sometimes reversibly associated with the membrane. However, due to their poor solubility, only a minority of integral membrane proteins have been identified, and most of them came out from in silico analyses since the genome of the model plant Arabidopsis thaliana was completed (2Arabidopsis Genome Initiative Analysis of the genome sequence of the flowering plant Arabidopsis thaliana..Nature. 2000; 408: 796-815Google Scholar). The Arabidopsis Membrane Protein Library (AMPL; www.biosci.cbs.umn.edu/Arabidopsis/) was established by clustering the predicted membrane proteins based on sequence and sorted into families of known, predicted, or unknown functions (3Ward J.M. Identification of novel families of membrane proteins from the model plant Arabidopsis thaliana..Bioinformatics. 2001; 17: 560-563Google Scholar). In ARAMEMNON, dedicated to Arabidopsis integral membrane proteins, averaging the predictions from seven publicly available programs led to the identification of ∼6,500 proteins displaying at least one transmembrane domain (TM) of the 25,500 predicted protein sequences (aramemnon.botanik.uni-koeln.de) (4Schwacke R. Schneider A. van der Graaff E. Fischer K. Catoni E. Desimone M. Frommer W.B. Flugge U.I. Kunze R. ARAMEMNON, a novel database for Arabidopsis integral membrane proteins..Plant Physiol. 2003; 131: 16-26Google Scholar). Some 1,800 of these proteins contain four TM or more and are possibly linked to transport functions. Among those, it is not possible to identify PM proteins because no signal peptide or specific signature specifying the targeting to PM have been identified so far. On the basis of two-dimensional (2-D) gel electrophoresis, Masson and Rossignol (5Masson F. Rossignol M. Basic plasticity of protein expression in tobacco leaf plasma membrane..Plant J. 1995; 8: 77-85Google Scholar) estimated that 500 polypeptides were present in the PM, corresponding to about 3% of total cellular proteins identified at that time. The total number of ∼750 PM proteins can now be inferred from the entire Arabidopsis proteome (2Arabidopsis Genome Initiative Analysis of the genome sequence of the flowering plant Arabidopsis thaliana..Nature. 2000; 408: 796-815Google Scholar). Nowadays, the real challenge is to find the way of extracting and identifying the whole set of PM proteins, including especially the integral proteins. Several methods have been developed on various animal and plant biological systems and already allowed identification of plant integral or PM-associated proteins. First, immunoscreening of a cDNA expression library with an antiserum raised against PM proteins was used to identify genes encoding PM proteins in soybean (6Shi J. Dixon R.A. Gonzales R.A. Kjellbom P. Bhattacharyya M.K. Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy..Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4457-4461Google Scholar) and A. thaliana (7Galaud J.P. Carriere M. Pauly N. Canut H. Chalon P. Caput D. Pont-Lezica R.F. Construction of two ordered cDNA libraries enriched in genes encoding plasmalemma and tonoplast proteins from a high-efficiency expression library..Plant J. 1999; 17: 111-118Google Scholar). Then, the sequence trap technique was designed to clone, in mammalian COS cells, cDNAs encoding secreted or membrane-associated proteins (8Kristoffersen P. Teichmann T. Stracke R. Palme K. Signal sequence trap to clone cDNAs encoding secreted or membrane-associated plant proteins..Anal. Biochem. 1996; 243: 127-132Google Scholar). However, the most commonly used technique for all organisms to identify new membrane proteins has been the solubilization of proteins from membrane-enriched fractions with detergents and their separation by 2-D gel electrophoresis (9Santoni V. Rouquie D. Doumas P. Mansion M. Boutry M. Degand H. Dupree P. Packman L. Sherrier J. Prime T. Bauw G. Posada E. Rouze P. Dehais P. Sahnoun I. Barlier I. Rossignol M. Use of a proteome strategy for tagging proteins present at the plasma membrane..Plant J. 1998; 16: 633-641Google Scholar, 10Rouquie D. Peltier J.B. Marquis-Mansion M. Tournaire C. Doumas P. Rossignol M. Construction of a directory of tobacco plasma membrane proteins by combined two-dimensional gel electrophoresis and protein sequencing..Electrophoresis. 1997; 18: 654-660Google Scholar, 11Molloy M.P. Herbert B.R. Walsh B.J. Tyler M.I. Traini M. Sanchez J.C. Hochstrasser D.F. Williams K.L. Gooley A.A. Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis..Electrophoresis. 1998; 19: 837-844Google Scholar, 12Simpson R.J. Connolly L.M. Eddes J.S. Pereira J.J. Moritz R.L. Reid G.E. Proteomic analysis of the human colon carcinoma cell line (LIM 1215): Development of a membrane protein database..Electrophoresis. 2000; 21: 1707-1732Google Scholar). Though numerous PM-specific proteins were identified by this method, most of these studies highlighted the fact that 2-D gel separation was not appropriate for a comprehensive mapping of membrane proteins. The first major problem is the chemical heterogeneity of proteins (isoelectric point (pI), molecular mass, and solubility). New 2-D PAGE procedures were developed to overcome this problem (13Gorg A. Boguth G. Obermaier C. Weiss W. Two-dimensional electrophoresis of proteins in an immobilized pH 4–12 gradient..Electrophoresis. 1998; 19: 1516-1519Google Scholar, 14Luche S. Santoni V. Rabilloud T. Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis..Proteomics. 2003; 3: 249-253Google Scholar). The second major limitation of this technique concerns protein solubility and/or hydrophobicity leading to an under-representation of the most hydrophobic proteins in 2-D gels (15Adessi C. Miege C. Albrieux C. Rabilloud T. Two-dimensional electrophoresis of membrane proteins: a current challenge for immobilized pH gradients..Electrophoresis. 1997; 18: 127-135Google Scholar, 16Wilkins M.R. Gasteiger E. Sanchez J.C. Bairoch A. Hochstrasser D.F. Two-dimensional gel electrophoresis for proteome projects: The effects of protein hydrophobicity and copy number..Electrophoresis. 1998; 19: 1501-1505Google Scholar). Indeed, such proteins often precipitate or aggregate during the isoelectric focusing electrophoresis. The solubility of membrane proteins in 2-D gels has been improved with the use of new zwitterionic detergents during sample preparation (17Chevallet M. Santoni V. Poinas A. Rouquie D. Fuchs A. Kieffer S. Rossignol M. Lunardi J. Garin J. Rabilloud T. New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis..Electrophoresis. 1998; 19: 1901-1909Google Scholar), and combined with the development of mass spectrometry analysis, many new proteins were identified in PM from bacteria (18le Coutre J. Whitelegge J.P. Gross A. Turk E. Wright E.M. Kaback H.R. Faull K.F. Proteomics on full-length membrane proteins using mass spectrometry..Biochemistry. 2000; 39: 4237-4242Google Scholar), human cells (1Harvey S. Zhang Y. Landry F. Miller C. Smith J.W. Insights into a plasma membrane signature..Physiol. Genomics. 2001; 5: 129-136Google Scholar), and Arabidopsis plants (19Santoni V. Kieffer S. Desclaux D. Masson F. Rabilloud T. Membrane proteomics: Use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties..Electrophoresis. 2000; 21: 3329-3344Google Scholar). However, the majority of proteins identified by this technique remains mainly peripheral proteins (20Santoni V. Molloy M. Rabilloud T. Membrane proteins and proteomics: Un amour impossible?.Electrophoresis. 2000; 21: 1054-1070Google Scholar). The last major problem is linked to the dynamics of protein expression in the cell. Low-abundance proteins, including regulatory proteins and rare membrane proteins, are out of the scope of standard proteomic techniques. One way to bring low-abundance proteins into view is to analyze subproteomes based on subcellular compartmentation (21Dreger M. Subcellular proteomics..Mass Spectrom. Rev. 2003; 22: 27-56Google Scholar). For instance, mammalian phagolysosome (22Garin J. Diez R. Kieffer S. Dermine J.F. Duclos S. Gagnon E. Sadoul R. Rondeau C. Desjardins M. The phagosome proteome: Insight into phagosome functions..J. Cell. Biol. 2001; 152: 165-180Google Scholar) and nuclear pore complex (23Cronshaw J.M. Krutchinsky A.N. Zhang W. Chait B.T. Matunis M.J. Proteomic analysis of the mammalian nuclear pore complex..J. Cell Biol. 2002; 158: 915-927Google Scholar), PM in plant cells (9Santoni V. Rouquie D. Doumas P. Mansion M. Boutry M. Degand H. Dupree P. Packman L. Sherrier J. Prime T. Bauw G. Posada E. Rouze P. Dehais P. Sahnoun I. Barlier I. Rossignol M. Use of a proteome strategy for tagging proteins present at the plasma membrane..Plant J. 1998; 16: 633-641Google Scholar, 24Santoni V. Vinh J. Pflieger D. Sommerer N. Maurel C. A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots..Biochem. J. 2003; 373: 289-296Google Scholar) or animal cells (1Harvey S. Zhang Y. Landry F. Miller C. Smith J.W. Insights into a plasma membrane signature..Physiol. Genomics. 2001; 5: 129-136Google Scholar), mitochondria from mammals (25Hanson B.J. Schulenberg B. Patton W.F. Capaldi R.A. A novel subfractionation approach for mitochondrial proteins: A three-dimensional mitochondrial proteome map..Electrophoresis. 2001; 22: 950-959Google Scholar) and plants (26Kruft V. Eubel H. Jansch L. Werhahn W. Braun H.P. Proteomic approach to identify novel mitochondrial proteins in Arabidopsis..Plant Physiol. 2001; 127: 1694-1710Google Scholar), and different chloroplast membranes (27Seigneurin-Berny D. Rolland N. Garin J. Joyard J. Technical Advance: Differential extraction of hydrophobic proteins from chloroplast envelope membranes: A subcellular-specific proteomic approach to identify rare intrinsic membrane proteins..Plant J. 1999; 19: 217-228Google Scholar, 28Peltier J.B. Friso G. Kalume D.E. Roepstorff P. Nilsson F. Adamska I. van Wijk K.J. Proteomics of the chloroplast: Systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins..Plant Cell. 2000; 12: 319-341Google Scholar, 29Ferro M. Salvi D. Riviere-Rolland H. Vermat T. Seigneurin-Berny D. Grunwald D. Garin J. Joyard J. Rolland N. Integral membrane proteins of the chloroplast envelope: Identification and subcellular localization of new transporters..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11487-11492Google Scholar). Complexity of the membrane fractions to be analyzed can be reduced using different strategies such as chloroform/methanol extraction (30Ferro M. Salvi D. Brugiere S. Miras S. Kowalski S. Louwagie M. Garin J. Joyard J. Rolland N. Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana..Mol. Cell. Proteomics. 2003; 2: 325-345Google Scholar), reverse-phase chromatography (31Gomez S.M. Nishio J.N. Faull K.F. Whitelegge J.P. The chloroplast grana proteome defined by intact mass measurements from liquid chromatography mass spectrometry..Mol. Cell. Proteomics. 2002; 1: 46-59Google Scholar), or blue natives gels (32Camacho-Carvajal M.M. Wollscheid B. Aebersold R. Steimle V. Schamel W.W. Two-dimensional blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: A proteomics approach..Mol. Cell. Proteomics. 2004; 3: 176-182Google Scholar). The aim of this work was to uncover new membrane proteins from Arabidopsis cell PM and especially new transport systems and ion channels. In addition, by focusing on proteins of the most intrinsic core of the membrane, which are difficult to pick up with classical proteomic approaches, we expected to complement previous studies of membrane proteins separated by 2-D PAGE. In this article, we report mass spectrometry analyses of proteins solubilized from PM fractions from Arabidopsis cell suspensions. As an informative subcellular proteomic approach requires highly purified subfractions to be obtained, the purity of PM prepared from Arabidopsis cell suspensions was systematically assessed. Then, proteins were extracted from PM using different procedures in order to retrieve proteins within a wide range of hydrophobicity, i.e. chloroform/methanol extraction and alkaline treatment. Mass spectrometry analyses (matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF), electrospray ionization (ESI) tandem mass spectrometry (MS/MS), and nano-liquid chromatography (LC)-MS/MS) led to the identification of 102 different proteins. Database annotations and predictions indicate that more than 50% of them are integral proteins with 1–12 TM domains. Most of these proteins had never been identified before in the course of 2-D gel analysis of Arabidopsis PM (33Santoni V. Doumas P. Rouquie D. Mansion M. Rabilloud T. Rossignol M. Large scale characterization of plant plasma membrane proteins..Biochimie. 1999; 81: 655-661Google Scholar). Moreover, PM localization of five new proteins was demonstrated by confocal microscopy. As to our objective, 34 proteins with at least four TM domains were identified, among which 20 proteins had been already characterized as transporters, and nine proteins display characteristics compatible with a transport function. A. thaliana cells were cultured in complete medium (34Jouanneau J.P. Péaud-Lenoël C. Croissance et synthèse des protéines de suspensions cellulaires de tabac sensibles à la kinétine..Physiol. Plantarum. 1967; 20: 834-850Google Scholar) under controlled conditions, continuous light, 23 °C, and 150 rotations per minute. Cells were collected after 5 days of culture, during the exponential phase. A microsomal fraction was obtained after grinding the cells and applying a series of differential centrifugations (35Canut H. Baudracco S. Cabané M. Boudet A.M. Marigo G. Preparation of sealed tonoplast and plasma-membrane vesicles from Catharanthus roseus (L.) G. Don. cells by free-flow electrophoresis..Planta. 1991; 184: 448-456Google Scholar). A PM-enriched fraction was purified from microsomes by the two-phase partitioning between polyethyleneglycol (PEG) and dextran (6.4% w/w) (36Larsson C. Widell S. Kjellbom P. Preparation of high-purity plasma membranes..Methods Enzymol. 1987; 148: 558-568Google Scholar). In this condition, the PEG upper phase is enriched in PM vesicles though the dextran lower phase, named endomembrane fraction, contains all other membranes. To eliminate contamination of the PM fraction with PEG, the PM fraction was again partitioned in a two-phases partition system consisting of 0.7 m PEG/K-PO4, pH 7 (37Busby T.F. Ingham K.C. Removal of polyethylene glycol from proteins by salt-induced phase separation..Vox Sang. 1980; 39: 93-100Google Scholar). In this system, PM is recovered in the saline lower phase. After an ultracentrifugation (110,000 × g), PM is recovered in the pellet. Proteins were resuspended in 50 mm 4-morpholinepropanesulfonic acid (MOPS)/NaOH, pH 7.8, 1 mm dithiothreitol (DTT). Protein amounts were estimated using Bradford procedure (38Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding..Anal. Biochem. 1976; 72: 248-254Google Scholar), and the proteins were concentrated at 10 mg/ml. The sensitivity of the Mg2+-ATPase activity to vanadate and KNO3 was used as a marker of PM and tonoplast, respectively (39Briskin D.P. Leonard R.T. Hodges T.K. Isolation of the plasma membrane: Membrane markers and general principles..Methods Enzymol. 1987; 148: 542-558Google Scholar). Cytochrome c oxidase activity was used as a marker of mitochondria (39Briskin D.P. Leonard R.T. Hodges T.K. Isolation of the plasma membrane: Membrane markers and general principles..Methods Enzymol. 1987; 148: 542-558Google Scholar). Hydrophobic proteins were extracted from the purified PM fraction using a C/M (v/v) treatment as described by Seigneurin-Berny et al. (27Seigneurin-Berny D. Rolland N. Garin J. Joyard J. Technical Advance: Differential extraction of hydrophobic proteins from chloroplast envelope membranes: A subcellular-specific proteomic approach to identify rare intrinsic membrane proteins..Plant J. 1999; 19: 217-228Google Scholar). For the standard conditions, 0.2 ml (2 mg) of PM fraction was slowly diluted in 1.8 ml of cold 5/4 C/M solution. The resulting mixture was stored for 15 min on ice before centrifugation (4 °C) for 20 min at 17,600 × g. Insoluble proteins in the organic phase were recovered as a white pellet at the bottom of the tube. The organic phase, which contains the proteins soluble in C/M solutions, was removed for further protein analyses. C/M-soluble proteins were dried under nitrogen, precipitated with acetone (80%), resuspended in 20 μl of SDS-PAGE buffer (4×), and stored at −80 °C. Before SDS-PAGE separation, the proteins were solubilized at 95 °C in Laemmli buffer. To analyze the effect of pH on the extraction efficiency, a 20 mm NaPi, pH 6 buffer was used instead of the classical 50 mm MOPS/NaOH, pH 7.8. To increase the diversity of extracted proteins, in some experiments the PM fraction was pretreated by 0.5% Triton X-100. After centrifugation (18,000 × g, 4 °C), the pellet and the supernatant were resuspended independently in the same volume and in the same buffer, leading to two subfractions, soluble and insoluble Triton (ST and IT). In that case, both ST and IT were used for C/M extraction. Denaturation at 37 °C before the SDS-PAGE was also tested, some proteins being damaged at 95 °C. The PM proteins (0.2 mg) were diluted in 0.2 ml of solubilization solution (50 mm MOPS/NaOH, pH 7.8, 1 mm DTT) containing either 1% (v/v) Triton X-100 or 0.1 m NaOH. After 30 min incubation on ice, the mixture was centrifuged (17,600 × g, 15 min, 4 °C) to separate two fractions: the supernatant containing proteins solubilized by the treatment and the pellet containing the insoluble proteins. The pellet proteins were then suspended in 50 μl of the initial solubilization solution (50 mm MOPS/NaOH, pH 7.8, 1 mm DTT) and both fractions were analyzed by SDS-PAGE as described above for C/M extracts. The different samples were separated on 10 or 12% acrylamide gels for SDS-PAGE analyses. Both gels (stacking and separation) and migration buffers contained 0.1% SDS. For some analyses, protein migration was stopped just between the stacking and the separating gels so that proteins were concentrated on a very thin band for further nanoLC-MS/MS analyses (30Ferro M. Salvi D. Brugiere S. Miras S. Kowalski S. Louwagie M. Garin J. Joyard J. Rolland N. Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana..Mol. Cell. Proteomics. 2003; 2: 325-345Google Scholar). Western blot analyses were performed after SDS-PAGE analysis of cell subfractions (microsome, endomembrane and PM fractions) as described by Maniatis et al. (40Maniatis T. Fritsh E.F. Sambrook J. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1982Google Scholar), each fraction contained 20 μg of proteins. Western blot experiments were performed with markers of different membrane compartments. The anti-H+-ATPase (P-type) antibody is raised against the PM H+-ATPase of Nicotiana plumbaginifolia (used at 1/250) (kindly provided by M. Boutry). The anti-E 37 antibody is raised against a protein from the inner envelope membrane of spinach chloroplast (used at 1/20,000). The anti-tonoplast intrinsic protein (TIP) antibody is directed against a tobacco tonoplast protein (used at 1/2,000) (kindly provided by C. Maurel and P. Gerbeau). The anti-Nad 9 antibody is raised against an extrinsic protein of the wheat mitochondrial inner membrane (used at 1/2,000), and the anti-TOM 40 antibody recognizes an outer membrane protein of yeast mitochondria (used at 1/2,000) (both kindly provided by J. M. Grienenberger and G. Bonnard). These antibodies were detected using alkaline phosphatase staining. The corresponding pre-immune sera were tested and gave no signal on blots. The in-gel digestion was carried out as previously described (41Ferro M. Seigneurin-Berny D. Rolland N. Chapel A. Salvi D. Garin J. Joyard J. Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins..Electrophoresis. 2000; 21: 3517-3526Google Scholar). Briefly, after separation by SDS-PAGE, individual bands were excised from the Coomassie blue-stained gel and washed with 50% acetonitrile and 25 mm NH4HCO3. Gel pieces were dried in a vacuum centrifuge and reswollen in 20 μl of 25 mm NH4HCO3 containing 0.5 μg of trypsin (sequencing grade; Promega, Madison, WI). After 4-h incubation at 37 °C, a 0.5-μl aliquot was removed for MALDI-TOF analysis and spotted onto the MALDI sample probe on top of a dried 0.5-μl mixture of 4 volumes solution of saturated ∝-cyano-4-hydroxy-trans-cinnamic acid in acetone and 3 volumes of nitrocellulose (10 mg/ml) dissolved in acetone/isopropanol 1/1 (v/v). Samples were rinsed by placing a 5-μl volume of 0.1% (v/v) trifluoroacetic acid on the matrix surface after the analyte solution had dried completely. After 2 min, the liquid was blown off by pressurized air. Tryptic digests were then subjected to MALDI-MS analysis on an Autoflex instrument (Bruker, Billerica, MA) in order to obtain peptide mass fingerprints. For protein identification purposes, mass spectrometric data were searched using the MS-Fit (prospector.ucsf.edu) or the Mascot softwares (www.matrixscience.com). Searching parameters were as follows: one missed cleavage, 100 ppm mass accuracy, six peptides allowed. For hits that did not fit with the tolerated mass accuracy and the occurrence of six peptides per protein, protein identification was achieved by MS/MS analysis. For ESI-MS/MS analyses, after in-gel tryptic digestion the gel pieces were then extracted with 5% (v/v) formic acid solution and acetonitrile. The extracts were combined with the original digest, and the sample was evaporated to dryness in vacuum centrifuge. The residues were dissolved in 0.1% (v/v) formic acid and desalted by using C18 Zip Tips (Millipore, Bedford, MA). Elution of the peptides was performed with 5–10 μl of a 50:50:0.1 (v/v) acetonitrile/H2O/formic acid solution. The peptide solution was introduced into a glass capillary (Protana, Odense, Denmark) for nanoESI. MS/MS experiments were carried out on a quadrupole TOF (QTOF) hybrid mass spectrometer (Waters, Micromass, Manchester, United Kingdom). Interpretation of MS/MS spectra was achieved manually and with the help of the PEPSEQ program (MassLynx software; Micromass). MS/MS sequence information was used for database searching by using either BLAST (www.ncbi.nlm.nih.gov/blast) or MS pattern (prospector.ucsf.edu) programs. For the LC-MS/MS, after the in-gel digestion gel the pieces were then extracted with 5% (v/v) formic acid solution and acetonitrile. After drying, tryptic peptides were resuspended in 0.5% aqueous trifluoroacetic acid. The samples were injected into a LC-Packings (Dionex, Sunnyvale, CA) or a CapLC (Waters) nanoLC system and first preconcentrated on a 300 μm × 5 mm PepMap C18 precolumn. The peptides were then eluted onto a C18 column (75 μm × 150 mm). The chromatographic separation used a gradient from solution A (5% acetonitrile:95% water:0.1% formic acid) to solution B (95% acetonitrile:5% water:0.1% formic acid) over 60 min at a flow rate of 200 nl/min. The LC system was directly coupled to QTOF1 or QTOF Ultima mass spectrometer (Waters). MS and MS/MS data were acquired and processed automatically using MassLynx 3.5 software. Database searching was carried out using the MASCOT 1.7 program available via internet. The parameters for Mascot searches were as followed: two missed cleavages; 2 and 0.4 Da mass accuracy allowed for parent and the fragment ions, respecti
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