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• W2018641191 abstract "Aquaporins form a family of water and solute channel proteins and are present in most living organisms. In plants, aquaporins play an important role in the regulation of root water transport in response to abiotic stresses. In this work, we investigated the role of phosphorylation of plasma membrane intrinsic protein (PIP) aquaporins in the Arabidopsis thaliana root by a combination of quantitative mass spectrometry and cellular biology approaches. A novel phosphoproteomics procedure that involves plasma membrane purification, phosphopeptide enrichment with TiO2 columns, and systematic mass spectrometry sequencing revealed multiple and adjacent phosphorylation sites in the C-terminal tail of several AtPIPs. Six of these sites had not been described previously. The phosphorylation of AtPIP2;1 at two C-terminal sites (Ser280 and Ser283) was monitored by an absolute quantification method and shown to be altered in response to treatments of plants by salt (NaCl) and hydrogen peroxide. The two treatments are known to strongly decrease the water permeability of Arabidopsis roots. To investigate a putative role of Ser280 and Ser283 phosphorylation in aquaporin subcellular trafficking, AtPIP2;1 forms mutated at either one of the two sites were fused to the green fluorescent protein and expressed in transgenic plants. Confocal microscopy analysis of these plants revealed that, in resting conditions, phosphorylation of Ser283 is necessary to target AtPIP2;1 to the plasma membrane. In addition, an NaCl treatment induced an intracellular accumulation of AtPIP2;1 by exerting specific actions onto AtPIP2;1 forms differing in their phosphorylation at Ser283 to induce their accumulation in distinct intracellular structures. Thus, the present study documents stress-induced quantitative changes in aquaporin phosphorylation and establishes for the first time a link with plant aquaporin subcellular localization. Aquaporins form a family of water and solute channel proteins and are present in most living organisms. In plants, aquaporins play an important role in the regulation of root water transport in response to abiotic stresses. In this work, we investigated the role of phosphorylation of plasma membrane intrinsic protein (PIP) aquaporins in the Arabidopsis thaliana root by a combination of quantitative mass spectrometry and cellular biology approaches. A novel phosphoproteomics procedure that involves plasma membrane purification, phosphopeptide enrichment with TiO2 columns, and systematic mass spectrometry sequencing revealed multiple and adjacent phosphorylation sites in the C-terminal tail of several AtPIPs. Six of these sites had not been described previously. The phosphorylation of AtPIP2;1 at two C-terminal sites (Ser280 and Ser283) was monitored by an absolute quantification method and shown to be altered in response to treatments of plants by salt (NaCl) and hydrogen peroxide. The two treatments are known to strongly decrease the water permeability of Arabidopsis roots. To investigate a putative role of Ser280 and Ser283 phosphorylation in aquaporin subcellular trafficking, AtPIP2;1 forms mutated at either one of the two sites were fused to the green fluorescent protein and expressed in transgenic plants. Confocal microscopy analysis of these plants revealed that, in resting conditions, phosphorylation of Ser283 is necessary to target AtPIP2;1 to the plasma membrane. In addition, an NaCl treatment induced an intracellular accumulation of AtPIP2;1 by exerting specific actions onto AtPIP2;1 forms differing in their phosphorylation at Ser283 to induce their accumulation in distinct intracellular structures. Thus, the present study documents stress-induced quantitative changes in aquaporin phosphorylation and establishes for the first time a link with plant aquaporin subcellular localization. Aquaporins form a family of channel proteins that mediate the transport across membranes of water, small neutral solutes, and occasionally ions (1Kaldenhoff R. Fischer M. Functional aquaporin diversity in plants.Biochim. Biophys. Acta. 2006; 1758: 1134-1141Crossref PubMed Scopus (163) Google Scholar, 2Maurel C. Plant aquaporins: novel functions and regulation properties.FEBS Lett. 2007; 581: 2227-2236Crossref PubMed Scopus (265) Google Scholar, 3Vandeleur R. Niemietz C.M. Tilbrook J. Tyerman S.D. Role of aquaporins in root responses to irrigation.Plant Soil. 2005; 274: 141-161Crossref Scopus (76) Google Scholar). Aquaporins are present in all living kingdoms and in plants. Aquaporins exhibit a characteristically high multiplicity of forms with for instance 35 members in Arabidopsis (4Johanson U. Karlsson M. Johansson I. Gustavsson S. Sjoüvall S. Fraysse L. Weig A.R. Kjellbom P. The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants.Plant Physiol. 2001; 126: 1-12Crossref Scopus (596) Google Scholar,5Quigley F. Rosenberg J.M. Shachar-Hill Y. Bohnert H.J. From genome to function: the Arabidopsis aquaporins.Genome Biol. 2002; 3: 1-17Google Scholar). Based upon their amino acid sequence homology, plant aquaporins can be classified into four subfamilies (4Johanson U. Karlsson M. Johansson I. Gustavsson S. Sjoüvall S. Fraysse L. Weig A.R. Kjellbom P. The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants.Plant Physiol. 2001; 126: 1-12Crossref Scopus (596) Google Scholar, 5Quigley F. Rosenberg J.M. Shachar-Hill Y. Bohnert H.J. From genome to function: the Arabidopsis aquaporins.Genome Biol. 2002; 3: 1-17Google Scholar, 6Zardoya R. Villalba S. A phylogenetic framework for the aquaporin family in eukaryotes.J. Mol. Evol. 2001; 52: 391-404Crossref PubMed Scopus (104) Google Scholar). One of these corresponds to the plasma membrane intrinsic proteins (PIPs). 1The abbreviations used are: PIP, plasma membrane intrinsic protein; GFP, green fluorescent protein; Lpr, root hydraulic conductivity; PM, plasma membrane; TiO2, titanium dioxide; WT, wild type; ER, endoplasmic reticulum. 1The abbreviations used are: PIP, plasma membrane intrinsic protein; GFP, green fluorescent protein; Lpr, root hydraulic conductivity; PM, plasma membrane; TiO2, titanium dioxide; WT, wild type; ER, endoplasmic reticulum. The PIPs with 13 members in Arabidopsis represent the most abundant aquaporins in the plasma membrane (PM) and can be further divided into two sequence homology groups (AtPIP1 and AtPIP2). Aquaporins are 25–35-kDa proteins that share a typical organization with six transmembrane α-helices interrupted by five connecting loops (loops A–E) (7Fujiyoshi Y. Mitsuoka K. de Groot B.L. Philippsen A. Grubmuller H. Agre P. Engel A. Structure and function of water channels.Curr. Opin. Struct. Biol. 2002; 12: 509-515Crossref PubMed Scopus (230) Google Scholar,8Tornroth-Horsefield S. Wang Y. Hedfalk K. Johanson U. Karlsson M. Tajkhorshid E. Neutze R. Kjellbom P. Structural mechanism of plant aquaporin gating.Nature. 2006; 439: 688-694Crossref PubMed Scopus (631) Google Scholar). In PM aquaporins, the N and C termini as well as loops B and D are exposed in the cytosol, whereas loops A, C, and E face the cell wall. Plants need to continuously adjust their water status in response to changing environmental conditions, and aquaporins play an important role in these processes (3Vandeleur R. Niemietz C.M. Tilbrook J. Tyerman S.D. Role of aquaporins in root responses to irrigation.Plant Soil. 2005; 274: 141-161Crossref Scopus (76) Google Scholar, 9Chaumont F. Moshelion M. Daniels M.J. Regulation of plant aquaporin activity.Biol. Cell. 2005; 97: 749-764Crossref PubMed Scopus (233) Google Scholar, 10Luu D.-T. Maurel C. Aquaporins in a challenging environment: molecular gears for adjusting plant water status.Plant Cell Environ. 2005; 28: 85-96Crossref Scopus (257) Google Scholar). In particular, physiological and genetics studies have provided compelling evidence for a role of aquaporins in the regulation, in response to abiotic stresses, of root water transport, i.e. root hydraulic conductivity (Lpr) (10Luu D.-T. Maurel C. Aquaporins in a challenging environment: molecular gears for adjusting plant water status.Plant Cell Environ. 2005; 28: 85-96Crossref Scopus (257) Google Scholar,11Javot H. Maurel C. The role of aquaporins in root water uptake.Ann. Bot. 2002; 90: 301-313Crossref PubMed Scopus (463) Google Scholar). For instance, exposure of Arabidopsis plants to salt (100 mm NaCl) induced a rapid (half-time, 45 min) and significant decrease (−70%) in Lpr that was maintained for at least 24 h (12Boursiac Y. Chen S. Luu D.-T. Sorieul M. van den Dries N. Maurel C. Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression.Plant Physiol. 2005; 139: 790-805Crossref PubMed Scopus (425) Google Scholar). Whereas the long term effect of this NaCl stress can be accounted for by an overall transcriptional down-regulation of aquaporins, the molecular mechanisms involved in the early inhibition of Lpr by NaCl are not fully understood yet. These mechanisms involve a slight decrease in overall abundance of AtPIP1 proteins as soon as 30 min after exposure to NaCl and a trafficking of AtPIP1 and AtPIP2 isoforms between the PM and intracellular compartments that may contribute to reducing the abundance of AtPIPs at the PM and therefore the hydraulic conductivity of salt-stressed root cells (12Boursiac Y. Chen S. Luu D.-T. Sorieul M. van den Dries N. Maurel C. Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression.Plant Physiol. 2005; 139: 790-805Crossref PubMed Scopus (425) Google Scholar). Chilling is another stress that leads to inhibition of Lpr, and a relationship between aquaporin regulation and reactive oxygen species was established in this context (13Lee S.H. Singh A.P. Chung G.C. Rapid accumulation of hydrogen peroxide in cucumber roots due to exposure to low temperature appears to mediate decreases in water transport.J. Exp. Bot. 2004; 55: 1733-1741Crossref PubMed Scopus (81) Google Scholar). In cucumber for instance, hydrogen peroxide (H2O2) accumulated in response to chilling, and treatment of roots with exogenous H2O2 inhibited Lpr to the same extent as chilling. In Arabidopsis a rapid decrease in Lpr can also be observed in response to 2 mm H2O2. 2Y. Boursiac, J. Boudet, O. Postaire, D.-T. Luu, C. Tournaire-Roux, and C. Maurel, submitted manuscript. 2Y. Boursiac, J. Boudet, O. Postaire, D.-T. Luu, C. Tournaire-Roux, and C. Maurel, submitted manuscript. Because of its amplitude (>70%) and rapidity (half-time, ≅8 min) this decrease is undoubtedly due to a down-regulation of root aquaporins. Post-translational modifications are central for regulating protein structure and function and thereby for modulating and controlling protein catalytic activity, subcellular localization, stability, and interaction with other partners. Qualitative and quantitative information about post-translational modifications and in particular measurements of their dynamic changes are now critically needed to understand the complexity of cell regulations. Protein phosphorylation is one of the most important and best characterized post-translational modifications. Virtually all cellular processes are regulated in one or multiple ways by reversible phosphorylation, and the identification of the protein kinases and phosphatases, their substrates, and the specific sites of phosphorylation involved is crucial for the understanding of cell signaling. Besides classical methods relying on in vivo and in vitro labeling or immunodetection of phosphorylated proteins, MS is now widely used for studies on protein phosphorylation (14Wolf-Yadlin A. Hautaniemi S. Lauffenburger D.A. White F.M. Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5860-5865Crossref PubMed Scopus (430) Google Scholar,15Jensen O.N. Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry.Curr. Opin. Chem. Biol. 2004; 8: 33-41Crossref PubMed Scopus (462) Google Scholar). Different instrumentations such as ESI- and MALDI-MS systems are now amenable to phosphoprotein analysis (16Bennett K.L. Stensballe A. Podtelejnikov A.V. Moniatte M. Jensen O.N. Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry.J. Mass Spectrom. 2002; 37: 179-190Crossref PubMed Scopus (97) Google Scholar), and sample preparation procedures have been optimized to enhance phosphopeptide recovery and detection by MS (17Stensballe A. Jensen O.N. Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides.Rapid Commun. Mass Spectrom. 2004; 18: 1721-1730Crossref PubMed Scopus (99) Google Scholar). In particular, immobilized metal affinity chromatography (18Zhou W. Merrick B.A. Khaledi M.G. Tomer K.B. Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry.J. Am. Soc. Mass Spectrom. 2000; 11: 273-282Crossref PubMed Scopus (170) Google Scholar) or titanium dioxide (TiO2) microcolumns (19Larsen M.R. Thingholm T.E. Jensen O.N. Roepstorff P. Jorgensen T.J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.Mol. Cell. Proteomics. 2005; 4: 873-886Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar) have proved powerful for the selective enrichment of phosphorylated peptides. Phosphorylated serine residues have been identified in the N-terminal and C-terminal tails of various plant aquaporins (20Daniel J.A. Yeager M. Phosphorylation of aquaporin PvTIP3;1 defined by mass spectrometry and molecular modeling.Biochemistry. 2005; 44: 14443-14454Crossref PubMed Scopus (37) Google Scholar, 21Guenther J.F. Chanmanivone N. Galetovic M.P. Wallace I.S. Cobb J.A. Roberts D.M. Phosphorylation of soybean nodulin 26 on serine 262 enhances water permeability and is regulated developmentally and by osmotic signals.Plant Cell. 2003; 15: 981-991Crossref PubMed Scopus (134) Google Scholar, 22Johansson I. Karlsson M. Shukla V.K. Chrispeels M.J. Larsson C. Kjellbom P. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation.Plant Cell. 1998; 10: 451-459Crossref PubMed Scopus (425) Google Scholar, 23Johansson I. Larsson C. Ek B. Kjellbom P. The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca2+ and apoplastic water potential.Plant Cell. 1996; 8: 1181-1191PubMed Google Scholar, 24Miao G.-H. Hong Z. Verma D.P.S. Topology and phosphorylation of soybean nodulin-26, an intrinsic protein of the peribacteroid membrane.J. Cell Biol. 1992; 118: 481-490Crossref PubMed Scopus (60) Google Scholar, 25Niittylaü T. Fuglsang A.T. Palmgren M.G. Frommer W.B. Schulze W.X. Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis.Mol. Cell. Proteomics. 2007; 6: 1711-1726Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). In particular, two phosphorylation sites were identified in the C terminus of Arabidopsis AtPIP2;1 (26Nuühse T.S. Stensballe A. Jensen O.N. Peck S.C. Phosphoproteomics of the Arabidopsisplasma membrane and a new phosphorylation site database.Plant Cell. 2004; 16: 2394-2405Crossref PubMed Scopus (406) Google Scholar) and AtPIP2;6 (25Niittylaü T. Fuglsang A.T. Palmgren M.G. Frommer W.B. Schulze W.X. Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis.Mol. Cell. Proteomics. 2007; 6: 1711-1726Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) and spinach SoPIP2;1 (22Johansson I. Karlsson M. Shukla V.K. Chrispeels M.J. Larsson C. Kjellbom P. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation.Plant Cell. 1998; 10: 451-459Crossref PubMed Scopus (425) Google Scholar) (Table I). AtPIP2;7 also shows double phosphorylation, but only one phosphosite was clearly identified (26Nuühse T.S. Stensballe A. Jensen O.N. Peck S.C. Phosphoproteomics of the Arabidopsisplasma membrane and a new phosphorylation site database.Plant Cell. 2004; 16: 2394-2405Crossref PubMed Scopus (406) Google Scholar) (see Table I). Also all plant AtPIPs show a conserved putative phosphorylation site in loop B (22Johansson I. Karlsson M. Shukla V.K. Chrispeels M.J. Larsson C. Kjellbom P. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation.Plant Cell. 1998; 10: 451-459Crossref PubMed Scopus (425) Google Scholar,23Johansson I. Larsson C. Ek B. Kjellbom P. The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca2+ and apoplastic water potential.Plant Cell. 1996; 8: 1181-1191PubMed Google Scholar). Based on functional analyses in Xenopus oocytes, it was proposed that phosphorylation of SoPIP2;1 at this site and at Ser262 (in the C terminus) was able to regulate its water transport activity (22Johansson I. Karlsson M. Shukla V.K. Chrispeels M.J. Larsson C. Kjellbom P. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation.Plant Cell. 1998; 10: 451-459Crossref PubMed Scopus (425) Google Scholar). A molecular mechanism for phosphorylation-dependent gating of PIPs has recently been proposed from the atomic structures of SoPIP2;1 in its open and closed conformations (8Tornroth-Horsefield S. Wang Y. Hedfalk K. Johanson U. Karlsson M. Tajkhorshid E. Neutze R. Kjellbom P. Structural mechanism of plant aquaporin gating.Nature. 2006; 439: 688-694Crossref PubMed Scopus (631) Google Scholar). Mammalian aquaporins also carry multiple phosphorylation sites, and by contrast to plant aquaporins, phosphorylation of mammalian aquaporin-2 is not involved in gating but rather regulates the shuttling of the protein between the PM and intracellular compartments (27Kamsteeg E.-J. Heijnen I. van Os C.H. Deen P.M.T. The subcellular localization of an aquaporin-2 tetramer depends on the stoichiometry of phosphorylated and non phosphorylated monomers.J. Cell Biol. 2000; 151: 919-929Crossref PubMed Scopus (147) Google Scholar,28Procino G. Carmosino M. Marin O. Brunanti A.M. Contri A. Pinna L. Mannucci R. Nielsen S. Kwon T.-H. Svelto M. Valenti G. Ser-256 phosphorylation dynamics of aquaporin 2 during maturation from the endoplasmic reticulum to the vesicular compartment in renal cells.FASEB J. 2003; 17: 1886-1910Crossref PubMed Scopus (74) Google Scholar).Table IPhosphorylation sites in the C-terminal tail of PIP aquaporinsProteinSequencen_phRef.AtPIP2;1/2;2/2;3277SLGpSFR282125Niittylaü T. Fuglsang A.T. Palmgren M.G. Frommer W.B. Schulze W.X. Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis.Mol. Cell. Proteomics. 2007; 6: 1711-1726Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 31Santoni 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: 289-296Crossref PubMed Scopus (118) Google ScholaraOne phosphorylation site was detected but not identified.AtPIP2;1/2;2/2;3277SLGpSFRSAANV2871Present workAtPIP2;1/2;2/2;3277SLGpSFRpSAANV287225Niittylaü T. Fuglsang A.T. Palmgren M.G. Frommer W.B. Schulze W.X. Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis.Mol. Cell. Proteomics. 2007; 6: 1711-1726Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 26Nuühse T.S. Stensballe A. Jensen O.N. Peck S.C. Phosphoproteomics of the Arabidopsisplasma membrane and a new phosphorylation site database.Plant Cell. 2004; 16: 2394-2405Crossref PubMed Scopus (406) Google Scholar, and present workAtPIP2;4277ALGpSFGSFGSFRSFA2911Present workAtPIP2;4277ALGpSFGpSFGSFRSFA2912Present workAtPIP2;4277ALGpSFGpSFGpSFRsFA2913Present workAtPIP2;4277ALGpSFGpSFGSFRpSFA2913Present workAtPIP2;6282pSQLHELHA289125Niittylaü T. Fuglsang A.T. Palmgren M.G. Frommer W.B. Schulze W.X. Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis.Mol. Cell. Proteomics. 2007; 6: 1711-1726Abstract Full Text Full Text PDF PubMed Scopus (213) Google ScholarAtPIP2;7270ALGpSFRSNATN2801Present workAtPIP2;7270ALGpSFRpSNATN280226Nuühse T.S. Stensballe A. Jensen O.N. Peck S.C. Phosphoproteomics of the Arabidopsisplasma membrane and a new phosphorylation site database.Plant Cell. 2004; 16: 2394-2405Crossref PubMed Scopus (406) Google Scholar, 34Hem S. Rofidal V. Sommerer N. Rossignol M. Novel subsets of the Arabidopsisplasmalemma phosphoproteome identify phosphorylation sites in secondary active transporters.Biochem. Biophys. Res. Commun. 2007; 363: 375-380Crossref PubMed Scopus (29) Google Scholar,bTwo phosphorylation sites were detected, but only Ser273 was identified as phosphorylated. and present workAtPIP2;7270ALGpSFRpSNApTN2803Present workSoPIP2;1271ALGpSFRSNPTN281122Johansson I. Karlsson M. Shukla V.K. Chrispeels M.J. Larsson C. Kjellbom P. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation.Plant Cell. 1998; 10: 451-459Crossref PubMed Scopus (425) Google ScholarSoPIP2;1271ALGpSFRpSNPTN281222Johansson I. Karlsson M. Shukla V.K. Chrispeels M.J. Larsson C. Kjellbom P. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation.Plant Cell. 1998; 10: 451-459Crossref PubMed Scopus (425) Google Scholara One phosphorylation site was detected but not identified.b Two phosphorylation sites were detected, but only Ser273 was identified as phosphorylated. Open table in a new tab The purpose of this work was to study the role of plant PM aquaporin phosphorylation in regulating the root water permeability in response to NaCl and H2O2 treatments. For this, a systematic inventory of phosphorylation sites in the C terminus of AtPIP aquaporins was performed, and novel phosphoresidues were discovered. Because of the emerging role of stimulus-dependent trafficking of plant aquaporins between the PM and intracellular compartments (12Boursiac Y. Chen S. Luu D.-T. Sorieul M. van den Dries N. Maurel C. Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression.Plant Physiol. 2005; 139: 790-805Crossref PubMed Scopus (425) Google Scholar, 29Barkla B.J. Vera-Estrella R. Pantoja O. Kirch H.-H. Bohnert H.J. Aquaporin localization—how valid are the TIP and PIP labels.Trends Plant Sci. 1999; 4: 86-88Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar,30Kirch H.H. Vera-Estrella R. Golldack D. Quigley F. Michalowski C.B. Barkla B.J. Bohnert H.J. Expression of water channel proteins in Mesembryanthemum crystallinum.Plant Physiol. 2000; 123: 111-124Crossref PubMed Scopus (145) Google Scholar), the role of aquaporin phosphorylation in this process was investigated. The results point to a specific phosphorylated site in the C terminus of AtPIP2;1 that regulates the trafficking of this aquaporin in control conditions and in response to an NaCl treatment. Endoproteinase Lys-C was purchased from Calbiochem. Synthetic PIP2 peptides (277SLGSFRSAANV297), either unmodified or singly phosphorylated at Ser280, were isotopically labeled on Arg281 with 13C and 15N to induce a 10-Da mass increment (Sigma). The same PIP2 peptide but diphosphorylated was isotopically labeled on Ala284 and Ala285 with 13C to induce a 6-Da mass increment (NeoMPS, Strasbourg, France). TiO2 beads were obtained by disassembling TiO2 guard columns purchased from GL Sciences Inc. (Tokyo, Japan). The 3M Empore™ C8 disks were from 3M Bioanalytical Technologies (St. Paul, MN). GELoader tips were from Eppendorf (Hamburg, Germany). 2,5-Hydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid were from Sigma-Aldrich. All other chemicals and reagents were of the highest commercially available grade. Arabidopsis thaliana ecotype Columbia (Col-0), plants were cultivated in hydroponic conditions as described previously (31Santoni 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: 289-296Crossref PubMed Scopus (118) Google Scholar). Briefly plants were cultivated in a growth chamber at 20 °C with an 8-h light (150 microeinstein m−2 s−1)/16-h dark cycle at 70% relative humidity. Plants were mounted on 35 × 35 × 0.6-cm polystyrene rafts floating in a basin filled with 8 liters of nutrient medium (1.25 mm KNO3, 0.75 mm MgSO4, 1.5 mm Ca(NO3)2, 0.5 mm KH2PO4, 0.1 mm Na2SiO3, 50 μm FeEDTA, 50 μm H3BO3, 12 μm MnSO4, 1 μm ZnSO4, 0.7 μm CuSO4, 0.24 μm MoO4Na2) and cultivated for up to 7 weeks. The effects of NaCl and H2O2 were then studied by complementing the nutrient solution with 100 mm NaCl for 2 and 4 h or 2 mm H2O2 for 15 min prior to root excision. Transgenic seedlings were cultivated for 8 days on half-strength Murashige and Skoog medium (32Murashige T. Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiol. Plant. 1962; 15: 473-497Crossref Scopus (52758) Google Scholar) without any antibiotic selection. The plantlets were then transferred for 2 or 4 h into a nutrient solution as described above complemented or not with 100 mm NaCl. A microsomal fraction was obtained from roots (31Santoni 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: 289-296Crossref PubMed Scopus (118) Google Scholar). Plasma membrane vesicles were purified by aqueous two-phase partitioning of the microsomal fraction in a mixture of polyethylene glycol 3350/dextran T-500, 6.4% (w/w) each in the presence of 5 mm KCl, as described previously (31Santoni 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: 289-296Crossref PubMed Scopus (118) Google Scholar). Protein concentration was measured using a modified Bradford procedure (31Santoni 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: 289-296Crossref PubMed Scopus (118) Google Scholar). The mean yield of PM extraction was 20 μg of protein/g of fresh weight. Extrinsic membrane proteins were stripped with a urea and NaOH treatment according to a previously described procedure (31Santoni 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: 289-296Crossref PubMed Scopus (118) Google Scholar). The abundance of AtPIP2 isoforms in PM samples was evaluated by an ELISA using an antibody raised against the last 17 amino acids of the AtPIP2;1 sequence as described previously (33Santoni V. Verdoucq L. Sommerer N. Vinh J. Pflieger D. Maurel C. Methylation of aquaporins in plant plasma membrane.Biochem. J. 2006; 400: 189-197Crossref PubMed Scopus (61) Google Scholar). The mean yield of AtPIP2 isoform was 5.3 pmol of PIP2/μg of PM proteins. Proteins were separated by SDS-PAGE on 12% acrylamide gels (31Santoni 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: 289-296Crossref PubMed Scopus (118) Google Scholar). The migrating band at 28 kDa was excised from SDS-PAGE and prepared for proteolytic digestion as described previously (31Santoni 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: 289-296Crossref PubMed Scopus (118) Google Scholar). Gel pieces containing 350 pmol of AtPIP2 aquaporins were reswollen in the presence of Lys-C at an enzyme:aquaporin ratio of 1:25 at 37 °C for 16 h. The supernatant of the digest was collected, and the remaining peptides were extracted in 0.1% TFA, 60% acetonitrile by sonication for 15 min. Supernatants were pooled, and the final volume was reduced to 10 μl using a centrifuge evaporator. To build up a TiO2 microcolumn, a small piece was stamped out of an Empore C8 disk by using a 200-μl pipette tip and placed at the constricted end of the GELoader tip, and TiO2 beads in suspension in acetonitrile were packed (34Hem S. Rofidal V. Sommerer N. Rossignol M. Novel subsets of the Arabidopsisplasmalemma phosphoproteome identify phosphorylation sites in secondary active transporters.Biochem. Biophys. Res. Commun. 2007; 363: 375-380Crossref PubMed Scopus (29) Google Scholar). The protein digest was then diluted in a loading buffer containing 80% acetonitrile and 0.1% TFA and loaded on the column, and the column was washed with 30 μl of loading buffer. Phosphopeptides were eluted with 3 μl of NH4OH at pH 12. 0.8 μl of eluted peptides was mixed with 0.8 μl of 20 mg/ml 2,5-hydroxybenzoic acid dissolved in acetonitrile, water, and phosphoric acid (50" @default.
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• W2018641191 creator A5046116452 @default.
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• W2018641191 date "2008-06-01" @default.
• W2018641191 modified "2023-10-17" @default.
• W2018641191 title "Multiple Phosphorylations in the C-terminal Tail of Plant Plasma Membrane Aquaporins" @default.
• W2018641191 cites W1546321334 @default.
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