title: In vivo phosphorylation sites of barley tonoplast proteins identified by a phosphoproteomic approach.
abstract: In plants the vacuolar functions are the cellular storage of soluble carbohydrates, organic acids, inorganic ions and toxic compounds. Transporters and channels located in the vacuolar membrane, the tonoplast, are modulated by PTMs to facilitate the optimal functioning of a large number of metabolic pathways. Here we present a phosphoproteomic approach for the identification of in vivo phosphorylation sites of tonoplast (vacuolar membrane) proteins. Highly purified tonoplast and tonoplast-enriched microsomes were isolated from photosynthetically induced barley (Hordeum vulgare) mesophyll protoplasts. Phosphopeptides were enriched by strong cation exchange (SCX) chromatography followed either by IMAC or titanium dioxide (TiO(2)) affinity chromatography and were subsequently analysed using LC-ESI-MS/MS. In total, 65 phosphopeptides of 27 known vacuolar membrane proteins were identified, including the two vacuolar proton pumps, aquaporins, CAX transporters, Na(+)/H(+) antiporters as well as other known vacuolar transporters mediating the transfer of potassium, sugars, sulphate and malate. The present study provides a novel source to further analyse the regulation of tonoplast proteins by protein phosphorylations, especially as most of the identified phosphorylation sites are highly conserved between Hordeum vulgare (Hv) and Arabidopsis thaliana.
authors.affiliation: Institute of Plant Biology, University of Zurich, Zurich, Switzerland. aendler@botinst.uzh.ch
authors.forename: Anne
authors.initials: A
authors.lastname: Endler
authors.forename: Sonja
authors.initials: S
authors.lastname: Reiland
authors.forename: Bertran
authors.initials: B
authors.lastname: Gerrits
authors.forename: Ulrike G
authors.initials: UG
authors.lastname: Schmidt
authors.forename: Sacha
authors.initials: S
authors.lastname: Baginsky
authors.forename: Enrico
authors.initials: E
authors.lastname: Martinoia
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D001467:Hordeum / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000737:chemistry; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D018515:Plant Leaves / Q000737:chemistry / Q000648:ultrastructure; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018087:Plastids / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics; D014617:Vacuoles / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D010748:Phosphopeptides; D010940:Plant Proteins