Novel subsets of the Arabidopsis plasmalemma phosphoproteome identify phosphorylation sites in secondary active transporters.

The generation of novel subsets of phosphorylation sites is needed to complement the present Arabidopsis plasma membrane phosphoprotein repertoire, where several families of proteins are under-represented. In this work, different combinations of chromatographic steps were first compared for capacity to resolve model phosphopeptides before characterisation from PSD fragments in MALDI MS/MS. Nearly half of the phosphorylation sites detected in the Arabidopsis plasmalemma using the optimised procedure were novel, and two-thirds of protein accessions identified secondary active transporters. These included phosphate/H(+) symporters, ammonium and nitrate transporters, different alkali cation exchangers, a urea/H(+) symporter, a glucose transporter, a purine permease, and peptide transporters. There has been previous functional evidence for phosphorylation of only a minority of these, the regulation of others having been essentially investigated at the transcriptional level. The demonstration of active phosphorylation sites in such a diverse set of secondary transporter families suggests that this regulation level plays a major role in the response of plants to nutrient availability.