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

液泡膜是一个重要的隔室，在各种环境条件下，溶质运输受到严格控制。所提出的实验的目标是通过以下实验阐明液泡膜蛋白磷酸化在调节液泡溶质积累和蛋白质靶向中的作用：（i）通过使用已知和预测的磷酸肽和相应的非磷酸肽的特异性标记的标准肽进行单反应监测的靶向分析将允许我们比较磷酸化状态和蛋白质丰度。多达20个关键转运蛋白和渠道在广泛的压力条件下将进行分析。(ii)将进行使用15N标记的非靶向“发现”方法，以鉴定特定应激条件或蛋白靶向途径突变导致亚细胞膜位置改变的蛋白。最后，TMT蛋白家族的不同磷酸化位点将被用作（iii）深入表征蛋白磷酸化在应激诱导的溶质积累和蛋白靶向中的作用的例子。

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.