Identification of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana

Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast‐enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform‐unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.

[1]  Wei-Hua Wu,et al.  A Protein Kinase, Interacting with Two Calcineurin B-like Proteins, Regulates K+ Transporter AKT1 in Arabidopsis , 2006, Cell.

[2]  K. Tomizawa,et al.  Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana. , 2004, Plant & cell physiology.

[3]  Alexandra ME Jones,et al.  Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses , 2007, The Plant journal : for cell and molecular biology.

[4]  B. Cubero,et al.  Alkali cation exchangers: roles in cellular homeostasis and stress tolerance. , 2006, Journal of experimental botany.

[5]  M. Fujita,et al.  Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments , 2004, Molecular Genetics and Genomics.

[6]  D. Bush,et al.  Protein phosphorylation plays a key role in sucrose-mediated transcriptional regulation of a phloem-specific proton–sucrose symporter , 2003, Planta.

[7]  K. Feldmann,et al.  TRH1 Encodes a Potassium Transporter Required for Tip Growth in Arabidopsis Root Hairs , 2001, Plant Cell.

[8]  T. Boller,et al.  Directed proteomics identifies a plant-specific protein rapidly phosphorylated in response to bacterial and fungal elicitors. , 2001, The Plant cell.

[9]  Rod B. Watson,et al.  Localization of Organelle Proteins by Isotope Tagging (LOPIT)*S , 2004, Molecular & Cellular Proteomics.

[10]  W. Snedden,et al.  Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. , 1999, Science.

[11]  T. Kuromori,et al.  Quantitative trait loci analysis of nitrate storage in Arabidopsis leading to an investigation of the contribution of the anion channel gene, AtCLC-c, to variation in nitrate levels. , 2004, Journal of experimental botany.

[12]  J. Garin,et al.  A Proteomics Dissection of Arabidopsis thaliana Vacuoles Isolated from Cell Culture*S , 2007, Molecular & Cellular Proteomics.

[13]  M. Mann,et al.  Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[14]  S. Peck,et al.  Phosphoproteomics in Arabidopsis: moving from empirical to predictive science. , 2006, Journal of experimental botany.

[15]  E. Martinoia,et al.  Transport processes of solutes across the vacuolar membrane of higher plants. , 2000, Plant & cell physiology.

[16]  J. Reed,et al.  A Mutation in the Arabidopsis KT2/KUP2 Potassium Transporter Gene Affects Shoot Cell Expansion Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010322. , 2002, The Plant Cell Online.

[17]  Joshua L. Heazlewood,et al.  SUBA: the Arabidopsis Subcellular Database , 2006, Nucleic Acids Res..

[18]  M. Mann,et al.  Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks , 2006, Cell.

[19]  S. Baginsky,et al.  Identification of a Vacuolar Sucrose Transporter in Barley and Arabidopsis Mesophyll Cells by a Tonoplast Proteomic Approach1 , 2006, Plant Physiology.

[20]  B. Ames.  ASSAY OF INORGANIC PHOSPHATE, TOTAL PHOSPHATE AND PHOSPHATASE , 1966 .

[21]  J. Calvete,et al.  The nitrate/nitrite ABC transporter of Phormidium laminosum: phosphorylation state of NrtA is not involved in its substrate binding activity. , 2006, Biochimica et biophysica acta.

[22]  A. Stensballe,et al.  Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry* , 2003, Molecular & Cellular Proteomics.

[23]  W. Toone,et al.  A novel pathway determining multidrug sensitivity in Schizosaccharomyces pombe , 2005, Genes to cells : devoted to molecular & cellular mechanisms.

[24]  J. Zhu,et al.  Plant salt tolerance. , 2001, Trends in plant science.

[25]  M. Rossignol.  Proteomic analysis of phosphorylated proteins. , 2006, Current opinion in plant biology.

[26]  D. Monachello,et al.  The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles , 2006, Nature.

[27]  Sirisha Aluri,et al.  Identification and functional expression of the Arabidopsis thaliana vacuolar glucose transporter 1 and its role in seed germination and flowering , 2007, Proceedings of the National Academy of Sciences.

[28]  F. Rubio,et al.  High-affinity potassium and sodium transport systems in plants. , 2006, Journal of experimental botany.

[29]  A. Hetherington,et al.  The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement , 2005, Nature.

[30]  Sung-ju Ahn,et al.  Expression of KT/KUP Genes in Arabidopsis and the Role of Root Hairs in K+ Uptake , 2004, Plant Physiology.

[31]  Sophie Alvarez,et al.  Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes , 2007, Proceedings of the National Academy of Sciences.

[32]  L. Looger,et al.  A cytosolic trans-activation domain essential for ammonium uptake , 2007, Nature.

[33]  T. Furukawa,et al.  Phosphorylation and functional regulation of CIC‐2 chloride channels expressed in Xenopus oocytes by M cyclin‐dependent protein kinase , 2002, The Journal of physiology.

[34]  Anthony Gobert,et al.  The two-pore channel TPK1 gene encodes the vacuolar K+ conductance and plays a role in K+ homeostasis , 2007, Proceedings of the National Academy of Sciences.

[35]  A. Stensballe,et al.  Phosphoproteomics of the Arabidopsis Plasma Membrane and a New Phosphorylation Site Databasew⃞ , 2004, The Plant Cell Online.

[36]  Thomas Girke,et al.  The Vegetative Vacuole Proteome of Arabidopsis thaliana Reveals Predicted and Unexpected Proteinsw⃞ , 2004, The Plant Cell Online.

[37]  M. Rossignol,et al.  Large‐scale characterization of integral proteins from Arabidopsis vacuolar membrane by two‐dimensional liquid chromatography , 2004, Proteomics.

[38]  U. Lüttge,et al.  The role of vacuolar malate-transport capacity in crassulacean acid metabolism and nitrate nutrition. Higher malate-transport capacity in ice plant after crassulacean acid metabolism-induction and in tobacco under nitrate nutrition. , 2000, Plant physiology.

[39]  Albert J R Heck,et al.  Quantitative Phosphoproteomics of Early Elicitor Signaling in Arabidopsis*S , 2007, Molecular & Cellular Proteomics.

[40]  D. J. Walker,et al.  Potassium homeostasis in vacuolate plant cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[41]  W. Frommer,et al.  Temporal Analysis of Sucrose-induced Phosphorylation Changes in Plasma Membrane Proteins of Arabidopsis*S , 2007, Molecular & Cellular Proteomics.

[42]  S. Baginsky,et al.  Novel Tonoplast Transporters Identified Using a Proteomic Approach with Vacuoles Isolated from Cauliflower Buds1[W][OA] , 2007, Plant Physiology.