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In vivo phosphorylation sites of barley tonoplast proteins identified by a phosphoproteomic approach
Author(s) -
Endler Anne,
Reiland Sonja,
Gerrits Bertran,
Schmidt Ulrike G.,
Baginsky Sacha,
Martinoia Enrico
Publication year - 2009
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200800323
Subject(s) - phosphorylation , in vivo , proteomics , biochemistry , biology , protein phosphorylation , microbiology and biotechnology , chemistry , gene , protein kinase a
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 .