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• W2076206711 abstract "The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered. The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered. The plasma membrane (PM) 1The abbreviations used are: PM, plasma membrane; GPI, glycosylphosphatidylinositol; TM, transmembrane; TAIR, The Arabidopsis Information Resource; NCBI, National Center for Biotechnology Information; PPDB, Plastid Proteome Database; ARF, ADP-ribosylation factor; LRR, leucine-rich repeat; RLK, receptor-like kinase; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; HIR, hypersensitive induced reaction protein; ER, endoplasmic reticulum; CYP, cyclophilin; SEC, Sec protein export pathway; RAB, Ras related in brain. has a peculiar status among membrane systems as it is directly in connection with the extracellular environment. Consequently this membrane is an essential element for cell primary functions, such as cellular differentiation or proliferation, and a privileged target for abiotic and biotic factors. To achieve all these functions, a large variety of proteins is necessary, including transport proteins, receptor proteins, and also proteins involved in signaling or cellular traffic. Schematically two types of membrane proteins occur. Integral proteins span the membrane and strongly interact with it usually through at least one hydrophobic transmembrane α-helix or less frequently with a β-barrel structure. Insertion of integral proteins into the membrane often needs the so-called secretion pathway through the endoplasmic reticulum and involves the use of an N-terminal signal peptide. When uncleaved by signal peptidase, the hydrophobic composition of the signal peptide can also participate in membrane binding. Peripheral proteins represent the second class of membrane proteins. They do not have typical hydrophobic domains and interact with the membrane with only one domain. Such an interaction may involve amphipathic helices, hydrophobic loops, and covalent links to lipid anchors, electrostatic interactions with the lipids of the membrane, or even protein-protein interactions. The investigation of a membrane proteome remains challenging with respect to both direct analyses (e.g. biochemical fractionation and proteomics analyses) and in silico approaches. Because of the existence of structural features such as the occurrence of a signal peptide or transmembrane (TM) α-helices, predictive analysis using bioinformatics has been a powerful means to predict membrane proteomes. For instance, 20–30% of the predicted ORFs of a typical animal or plant proteome are usually predicted to display at least one TM helix (1Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9290) Google Scholar). A combination of various criteria, based on both the amino acid sequence of an ORF including prediction of endoplasmic targeting and the occurrence of TM helices, led to the proposal that as much as 25% of the ORFs of the plant Arabidopsis thaliana corresponded to potential integral membrane proteins (2Schwacke R. Schneider A. van der Graaff E. Fischer K. Catoni E. Desimone M. Frommer W.B. Flugge U. Kunze R. ARAMEMNON, a novel database for Arabidopsis integral membrane proteins.Plant Physiol. 2003; 131: 16-26Crossref PubMed Scopus (526) Google Scholar). Prediction of the exact membrane or compartment where these proteins are targeted is a hard task as well and often leads to confusing or contradictive interpretations (3Heazlewood J.L. Tonti-Filippini J. Verboom R.E. Millar A.H. Combining experimental and predicted datasets for determination of the subcellular location of proteins in Arabidopsis.Plant Physiol. 2005; 139: 598-609Crossref PubMed Scopus (107) Google Scholar). Identification of the peripheral membrane proteins is a much more problematic issue. Whole genome predictions of lipid modification by N-myristoylation, prenylation, or glycosylphosphatidylinositol (GPI) anchors have been initiated as an attempt toward the identification of peripheral proteomes (4Maurer-Stroh S. Eisenhaber B. Eisenhaber F. N-terminal N-myristoylation of proteins: prediction of substrate proteins from amino acid sequence.J. Mol. Biol. 2002; 317: 541-557Crossref PubMed Scopus (192) Google Scholar, 5Boisson B. Giglione C. Meinnel T. Unexpected protein families including cell defense components feature in the N-myristoylome of a higher eukaryote.J. Biol. Chem. 2003; 278: 43418-43429Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 6Galichet A. Gruissem W. Protein farnesylation in plants—conserved mechanisms but different targets.Curr. Opin. Plant Biol. 2003; 6: 530-535Crossref PubMed Scopus (61) Google Scholar, 7Borner G.H.H. Sherrier D.J. Stevens T.J. Arkin I.T. Dupree P. Prediction of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A genomic analysis.Plant Physiol. 2002; 129: 486-499Crossref PubMed Scopus (159) Google Scholar). The ratio of peripheral proteins modified by lipids is assessed to be less than 4%, and the issue of the exact membrane location is not yet solved. Because they do not have specific features in their amino acid sequence, other types of peripheral membrane proteins are difficult to identify through predictive approaches. To make a decisive contribution to the remaining unanswered issues relative to membrane localization and identification of peripheral proteins, direct approaches involving membrane fractionation and protein characterization by proteomics investigation are needed. Such approaches are fully expected also to confirm the predictions. Due to their diversity of functions, the PM proteins display a wide variety of biochemical properties, and they are associated with lipid domains differing in composition. Consequently a combination of techniques that differentially fractionates proteins according to their physicochemical properties and/or their degree of their integration in the membrane is expected to allow a better overview of the PM proteome protein diversity. Early attempts have led to the identification of less than 300 proteins as part of the PM proteome, i.e. 1 order of magnitude less than the value expected from the aforementioned bioinformatics studies (8Santoni 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: 633-641Crossref PubMed Google Scholar, 9Marmagne A. Rouet M. Ferro M. Rolland N. Alcon C. Joyard J. Garin J. Barbier-Brygoo H. Ephritikhine G. Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.Mol. Cell. Proteomics. 2004; 3: 675-691Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 10Alexandersson E. Saalbach G. Larsson C. Kjellbom P. Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking.Plant Cell Physiol. 2004; 45: 1543-1556Crossref PubMed Scopus (209) Google Scholar, 11Dunkley T.P.J. Hester S. Shadforth I.P. Runions J. Weimar T. Hanton S.L. Griffin J.L. Bessant C. Brandizzi F. Hawes C. Watson R.B. Dupree P. Lilley K.S. Mapping the Arabidopsis organelle proteome.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6518-6523Crossref PubMed Scopus (443) Google Scholar). More targeted plant plasma membrane analyses provided a more extensive coverage of the plasma membrane proteome including some GPI-anchored and lipid raft proteins (12Borner G.H.H. Sherrier D.J. Weimar T. Michaelson L.V. Hawkins N.D. Macaskill A. Napier J.A. Beale M.H. Lilley K.S. Dupree P. Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts.Plant Physiol. 2005; 137: 104-116Crossref PubMed Scopus (388) Google Scholar, 13Mongrand S. Morel J. Laroche J. Claverol S. Carde J. Hartmann M. Bonneu M. Simon-Plas F. Lessire R. Bessoule J. Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane.J. Biol. Chem. 2004; 279: 36277-36286Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 14Elortza F. Mohammed S. Bunkenborg J. Foster L.J. Nuhse T.S. Brodbeck U. Peck S.C. Jensen O.N. Modification-specific proteomics of plasma membrane proteins: identification and characterization of glycosylphosphatidylinositol-anchored proteins released upon phospholipase D treatment.J. Proteome Res. 2006; 5: 935-943Crossref PubMed Scopus (105) Google Scholar, 15Elortza F. Nuhse T.S. Foster L.J. Stensballe A. Peck S.C. Jensen O.N. Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins.Mol. Cell. Proteomics. 2003; 2: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 16Morel J. Claverol S. Mongrand S. Furt F. Fromentin J. Bessoule J. Blein J. Simon-Plas F. Proteomics of plant detergent-resistant membranes.Mol. Cell. Proteomics. 2006; 5: 1396-1411Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Collectively a set of about 500 PM proteins, identified via proteomics analyses, has been reported in the literature so far, indicating that a majority of PM proteins are still to be identified through such approaches. In a previous study, about 100 putative proteins were identified as part of the hydrophobic plasma membrane proteome isolated from Arabidopsis cell suspensions, 95% of which were yet unidentified proteins (9Marmagne A. Rouet M. Ferro M. Rolland N. Alcon C. Joyard J. Garin J. Barbier-Brygoo H. Ephritikhine G. Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.Mol. Cell. Proteomics. 2004; 3: 675-691Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). To increase the PM repertoire, a highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts; the insoluble fractions were analyzed by nano-LC-MS/MS mass spectrometry. From these two approaches, we identified around 450 proteins, among which more than 45% were predicted to possess transmembrane domains and/or to be anchored to the membrane. Moreover among these 450 proteins, we identified 289 proteins (65%) that had never been reported in other plant PM proteomics investigations. In the present report, we describe the largest plasma membrane proteome reported so far, allowing us to give a deeper insight into the plant PM and its role in the cell. The Arabidopsis cell culture condition, the plasma membrane purification procedure, and associated purity assessments were as described in Marmagne et al. (9Marmagne A. Rouet M. Ferro M. Rolland N. Alcon C. Joyard J. Garin J. Barbier-Brygoo H. Ephritikhine G. Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.Mol. Cell. Proteomics. 2004; 3: 675-691Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 17Marmagne A. Salvi D. Rolland N. Ephritikhine G. Joyard J. Barbier-Brygoo H. Purification and fractionation of membranes for proteomic analyses.Methods Mol. Biol. 2006; 323: 403-420PubMed Google Scholar). Briefly 5-day-old suspension culture cells were collected, and a microsomal fraction was obtained after grinding and a series of differential centrifugations. A PM-enriched fraction was purified from microsomes by two-phase partitioning between polyethylene glycol and dextran (6.4%, w/w). To eliminate polyethylene glycol, a second partitioning was achieved with a 0.7 m KH2PO4 solution. The PM fraction recovered in the saline lower phase was ultracentrifuged (110,000 × g), and the resulting pellet was resuspended in 50 mm MOPS/NaOH (pH 7.8), 1 mm dithiothreitol. This yielded the purified PM fraction that was further analyzed. Preparations of NaCl- and Na2CO3-insoluble protein fractions were as described previously (17Marmagne A. Salvi D. Rolland N. Ephritikhine G. Joyard J. Barbier-Brygoo H. Purification and fractionation of membranes for proteomic analyses.Methods Mol. Biol. 2006; 323: 403-420PubMed Google Scholar). PM protein fractions (0.2 mg) were suspended in 0.2 ml (final volume) of 1 m NaCl or 0.1 m Na2CO3. Each mixture was chilled for 15–30 min on ice and centrifuged for 20 min at 15,000 × g. Insoluble proteins were recovered as pellets and resuspended in denaturing gel electrophoresis buffer (8% SDS (v/v), 0.188 m Tris-HCl (pH 6.8), 0.1% bromphenol blue (v/v), 0.16 m dithiothreitol, 40% glycerol (v/v)). One-third of either the NaCl or Na2CO3 PM fraction was resolved by 12% SDS-PAGE. Sample migration was allowed for a length of 3.5 cm according to bromphenol blue stain. After Coomassie Blue staining of each salt fraction (NaCl and Na2CO3, respectively), the gel was cut into 11 discrete bands. In-gel digestion with trypsin (sequencing grade; Promega, Madison, WI) as proteolytic enzyme was carried out as described previously (9Marmagne A. Rouet M. Ferro M. Rolland N. Alcon C. Joyard J. Garin J. Barbier-Brygoo H. Ephritikhine G. Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.Mol. Cell. Proteomics. 2004; 3: 675-691Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) with the following modification. After washing and drying, gel pieces were rehydrated in 100 μl of 7% H2O2 at room temperature for 15 min in the dark. This step led to cysteine oxidation and conversion of the methionine residues into sulfone (18Jaquinod M. Villiers F. Kieffer-Jaquinod S. Hugouvieux V. Bruley C. Garin J. Bourguignon J. A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture.Mol. Cell. Proteomics. 2007; 6: 394-412Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Gel pieces were then finally extracted with 5% (v/v) formic acid solution. Tryptic peptides were resuspended in 0.5% aqueous trifluoroacetic acid. The samples were injected into a CapLC (Waters) nano-LC 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 (2% acetonitrile, 98% water, 0.1% formic acid) to solution B (80% acetonitrile, 20% water, 0.08% formic acid) over 60 min at a flow rate of 200 nl/min. The LC system was directly coupled to a Q-TOF Ultima mass spectrometer (Waters). MS and MS/MS data were acquired and processed automatically using MassLynx 4.0 software. Mascot was used for database searching, and proteins that were identified with at least two peptides both showing a score higher than 40 were automatically validated. The score threshold for automatic validation was fixed at 40 because (i) for both NaCl and Na2CO3 datasets, this value is just above the Mascot score above which there is identity or extensive homology for peptide assignment with a probability of at least 95% as quoted by the Mascot output and (ii) the false positive rate, as described by Peng et al. (19Peng J. Elias J.E. Thoreen C.C. Licklider L.J. Gygi S.P. Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome.J. Proteome Res. 2003; 2: 43-50Crossref PubMed Scopus (1388) Google Scholar), was estimated to be 0.1 and 0% for the NaCl and the Na2CO3 datasets, respectively. Consequently we estimated that proteins identified by at least two peptides with a score higher than 40 were significantly present in our samples. For proteins identified by only one peptide having a score higher than 40, the peptide sequence was checked manually. Peptides with scores higher than 20 and lower than 40 were systematically checked and/or interpreted manually to confirm or cancel the Mascot suggestion. Such a validation and false positive rate assessment was achieved using a home-made validation software, which allowed protein redundancy to be eliminated on the basis of protein identification by the same set or a subset of peptides (see Supplemental Table III) from both NaCl and Na2CO3 datasets. Database searching was carried out using the Mascot 2.0 program (Matrix Science). Two databases were used: a home-made list of well known contaminants (keratins and trypsin; 21 entries) and an updated compilation of the A. thaliana protein database provided by TAIR (nuclear, mitochondrial, and chloroplastic genome; TAIR version 6.0; July 9, 2006; 30,899 entries). The variable modifications allowed were as follows: acetyl (protein), acetyl N terminus, methionine oxidation, methionine sulfone, cysteic acid, false mass assignment + 1, and false mass assignment + 2. Two missed trypsin cleavages were allowed. Similarly to other plasma membrane proteomes, about 30 ribosomal proteins were identified (60 and 40 S) that likely originated from cytoskeleton-bound polysomes anchored to the plasma membrane via actin filaments (20Hesketh J.E. Sorting of messenger RNAs in the cytoplasm: mRNA localization and the cytoskeleton.Exp. Cell Res. 1996; 225: 219-236Crossref PubMed Scopus (91) Google Scholar, 21Medalia O. Weber I. Frangakis A.S. Nicastro D. Gerisch G. Baumeister W. Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography.Science. 2002; 298: 1209-1213Crossref PubMed Scopus (674) Google Scholar). A total of 446 proteins could eventually be retrieved. Protein names and/or associated functions were retrieved from NCBI (www.ncbi.nlm.nih.gov/entrez), PPDB (ppdb.tc.cornell.edu/), or TAIR (www.arabidopsis.org). GRAVY, molecular weight, pI, and TargetP (22Emanuelsson O. Nielsen H. Brunak S. von Heijne G. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence.J. Mol. Biol. 2000; 300: 1005-1016Crossref PubMed Scopus (3638) Google Scholar) annotations were retrieved from PPDB (ppdb.tc.cornell.edu/) (23Friso G. Giacomelli L. Ytterberg A.J. Peltier J. Rudella A. Sun Q. Wijk K.J.V. In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database.Plant Cell. 2004; 16: 478-499Crossref PubMed Scopus (386) Google Scholar). Predictions of membrane-spanning regions (i.e. transmembrane domains) were collected from the ARAMEMNON database (2Schwacke R. Schneider A. van der Graaff E. Fischer K. Catoni E. Desimone M. Frommer W.B. Flugge U. Kunze R. ARAMEMNON, a novel database for Arabidopsis integral membrane proteins.Plant Physiol. 2003; 131: 16-26Crossref PubMed Scopus (526) Google Scholar). The HMMTOP program (24Tusnady G.E. Simon I. The HMMTOP transmembrane topology prediction server.Bioinformatics. 2001; 17: 849-850Crossref PubMed Scopus (1580) Google Scholar) was also used for TM domain predictions. Post-translational modification predictions were performed on line using various dedicated programs. N-terminal myristoylation was first predicted by TermiNator (25Frottin F. Martinez A. Peynot P. Mitra S. Holz R.C. Giglione C. Meinnel T. The proteomics of N-terminal methionine cleavage.Mol. Cell. Proteomics. 2006; 5: 2336-2349Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar) using relaxed criteria. Positive entries were next validated using the updated (December 2006) version of the Arabidopsis database of myristoylated proteins (25Frottin F. Martinez A. Peynot P. Mitra S. Holz R.C. Giglione C. Meinnel T. The proteomics of N-terminal methionine cleavage.Mol. Cell. Proteomics. 2006; 5: 2336-2349Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar) using the software developed by Boisson et al. (5Boisson B. Giglione C. Meinnel T. Unexpected protein families including cell defense components feature in the N-myristoylome of a higher eukaryote.J. Biol. Chem. 2003; 278: 43418-43429Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). S-Palmitoylation on vicinal cysteines next to the N terminus of N-myristoylated proteins was predicted with TermiNator (26Meinnel T. Peynot P. Giglione C. Processed N-termini of mature proteins in higher eukaryotes and their major contribution to dynamic proteomics.Biochimie (Paris). 2005; 87: 701-712Crossref PubMed Scopus (66) Google Scholar). C-terminal prenylation was predicted by PrePS (27Maurer-Stroh S. Eisenhaber F. Refinement and prediction of protein prenylation motifs.Genome Biol. 2005; 6R: 55Crossref Google Scholar). Robust predictions of GPI anchors were retrieved from the ARAMEMNON database that compiles three prediction programs (big-PI, DGPI, and GPI-SOM). PM enrichment and degree of purity were assessed as described in Marmagne et al. (9Marmagne A. Rouet M. Ferro M. Rolland N. Alcon C. Joyard J. Garin J. Barbier-Brygoo H. Ephritikhine G. Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.Mol. Cell. Proteomics. 2004; 3: 675-691Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) on each new PM preparation by Western blots (data not shown). Considering previous biochemical and immunological tests, chloroplastic and mitochondrial contamination was estimated to be less than 5% each. Salt treatments (including NaCl and Na2CO3) of the membrane fraction are believed to abolish electrostatic interactions with integral membrane proteins or the polar head of lipids (28Ephritikhine G. Ferro M. Rolland N. Plant membrane proteomics.Plant Physiol. Biochem. 2004; 42: 943-962Crossref PubMed Scopus (77) Google Scholar). Improperly or weakly linked membrane proteins are eliminated by such treatments, leading to an enrichment in genuine membrane proteins and to a lower sample complexity. Sodium chloride and sodium carbonate, which have been proven to be efficient in previous analyses (29Santoni V. Doumas P. Rouquie D. Mansion M. Rabilloud T. Rossignol M. Large scale characterization of plant plasma membrane proteins.Biochimie (Paris). 1999; 81: 655-661Crossref PubMed Scopus (58) Google Scholar, 30Rolland N. Ferro M. Ephritikhine G. Marmagne A. Ramus C. Brugiere S. Salvi D. Seigneurin-Berny D. Bourguignon J. Barbier-Brygoo H. Joyard J. Garin J. A versatile method for deciphering plant membrane proteomes.J. Exp. Bot. 2006; 57: 1579-1589Crossref PubMed Scopus (25) Google Scholar), were chosen to increase further our knowledge of the PM proteome. Analyses of insoluble NaCl and Na2CO3 plasma membrane fractions led to the identification of 446 proteins (356 and 243, respectively) including only 153 common entries (34%). In this set, only 42 entries (9%) have already been identified in the hydrophobic PM proteome resulting from a chloroform/methanol solubilization or an alkaline washing of the PM fraction (9Marmagne A. Rouet M. Ferro M. Rolland N. Alcon C. Joyard J. Garin J. Barbier-Brygoo H. Ephritikhine G. Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.Mol. Cell. Proteomics. 2004; 3: 675-691Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) demonstrating that the different treatments are complementary. To classify the 446 proteins (Supplemental Table I), we compiled (i) information about the proteomics data (number of peptides, etc.), (ii) results of predictions by bioinformatics tools (TM domains, cell localization, etc.), (iii) information (function, localization, etc.) retrieved from different protein databases (TAIR, NCBI, and ExPaSy), and (iv) appropriate literature as indicated in Supplemental Table I. Table I shows that 286 of 446 proteins had no predicted subcellular targeting. 110 proteins were predicted as secreted proteins, strongly suggesting PM association (Table I). Indeed 85 of these 446 proteins have been experimentally shown to be localized in membranes in particular 61 in PM (Supplemental Table I). Among the 26 proteins targeted to mitochondria, 10 have already been identified in other PM proteomes, and three have been shown to be localized in the PM, suggesting an inaccurate targeting prediction, for at least part of them, unless these proteins have a dual targeting (Table I). For example, the small G-proteins ARFs were predicted to be mitochondrial, although they are known to be involved in the secretory pathway and cytoskeleton organization (31Vernoud V. Horton A.C. Yang Z. Nielsen E. Analysis of the small GTPase gene superfamily of Arabidopsis.Plant Physiol. 2003; 131: 1191-1208Crossref PubMed Scopus (505) Google Scholar). The occurrence of a PM receptor for GDP-bound Arf6 in mammal cells (32Klein S. Franco M. Chardin P. Luton F. Role of the Arf6 GDP/GTP cycle and Arf6 GTPase-activating proteins in actin remodeling and intracellular transport.J. Biol. Chem. 2006; 281: 12352-12361Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) is in agreement with a PM localization of ARFs. Likewise prediction of a mitochondrial location for V-ATPase subunits is likely to be wrong because the associated function is usually assigned to the tonoplast and/or the PM (33Nishi T. Forgac M. The vacuolar (H+)-ATPases—nature's most versatile proton pumps.Nat. Rev. Mol. Cell Biol. 2002; 3: 94-103Crossref PubMed Scopus (1005) Google Scholar, 34Rouquie D. Tournaire-Roux C. Szponarski W. Rossignol M. Doumas P. Cloning of the V-ATPase subunit G in plant: functional expression and sub-cellular localization.FEBS Lett. 1998; 437: 287-292Crossref PubMed Scopus (32) Google Scholar, 35Toyomura T. Murata Y. Yamamoto A. Oka T. Sun-Wada G. Wada Y. Futai M. From lysosomes to the plasma membrane: localization of vacuolar-type H+-ATPase with the a3 isoform during osteoclast differentiation.J. Biol. Chem. 2003; 278: 22023-22030Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Wrong predictions were also found among proteins predicted to be chloroplastic (Table I). This appears to be the case of newly identified plasma membrane proteins, such as CDPK7 (36Dammann C. Ichida A. Hong B. Romanowsky S.M. Hrabak E.M. Harmon A.C. Pickard B.G. Harper J.F. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis.Plant Physiol. 2003; 132: 1840-1848Crossref PubMed Scopus (218) Google Scholar), PHOT2 (37Kong S. Suzuki T. Tamura K. Mochizuki N. Hara-Nishimura I. Nagatani A. Blue light-induced association of phototropin 2 with the Golgi apparatus.Plant J. 2006; 45: 994-1005Crossref PubMed Scopus (123) Google Scholar), or PDR8 (38Kobae Y. Sekino T. Yoshioka H. Nakagawa T. Martinoia E. Maeshima M. Loss of AtPDR8, a plasma membrane ABC transporter of Arabidopsis thaliana, causes hypersensitive cell death upon pathogen infection.Plant Cell Physiol. 2006; 47: 309-318Crossref PubMed Scopus (135) Google Scholar). Such dubious chloroplastic predictions may involve at least five proteins that have been found in previous PM proteomes (Table I and Supplemental Table I). In this context, it is noteworthy that abundant mitochondrial and chloroplastic membrane proteins, such as the ATP synthase subunits (39Brugiere S. Kowalski S. Ferro M. Seigneurin-Berny D. Miras S. Salvi D. Ravanel S. d'Herin P. Garin J. Bourguignon J. Joyard J. Rolland N. The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions.Phytochemistry. 2004; 65: 1693-1707Crossref PubMed Scopus (119) Google Scholar) and the triose phosphate translocator (40Ferro 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 thali" @default.
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• W2076206711 date "2007-11-01" @default.
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• W2076206711 title "A High Content in Lipid-modified Peripheral Proteins and Integral Receptor Kinases Features in the Arabidopsis Plasma Membrane Proteome" @default.
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