The role of tonoplast protein phosphorylation in activity regulation and protein targeting

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The tonoplast membrane is an important compartment across which solute transport is tightly controlled under various environmental conditions. The goal of the proposed experiments is to elucidate the role of tonoplast protein phosphorylation in regulation of vacuolar solute accumulation and in protein targeting by the following experiments: (i) A targeted analysis by single reaction monitoring using specific labelled standard peptides of already known and predicted phosphopeptides and respective non-phosphopeptides will allow us to compare phosphorylation status and protein abundance. Up to 20 key transporters and channels across a wide range of stress conditions will be analyzed. (ii) An untargeted “discovery” approach using 15N-labeing will be carried out to identify proteins for which specific stress conditions or mutations of protein targeting pathways result in altered subcellular membrane location. Finally, different phosphorylation sites of the TMT protein family will be used as an example for (iii) in-depth characterization of the role of protein phosphorylation in stress-induced solute accumulation and protein targeting.