Identification of tonoplast membrane proteins involved in cellular response to environmental stress by quantitative/comparative- and phospho-proteomics

蛋白质组分析代表了一个强大的工具，以确定空泡蛋白和启发其相关性在应激反应。在过去的研究资助期间，我们主要致力于建立一个平台来分离高纯度的液泡，适用于基于质谱的蛋白质组学。我们从冷培养和对照植物中分离液泡，研究冷驯化过程中叶绿体蛋白质组的变化。因此，细胞质蛋白丰度的一些变化和蛋白磷酸化的改变被确定，为液泡在冷驯化中的生理相关性提供了第一个见解。通过这些蛋白质组分析，我们确定了一些未知的蛋白质，并对其中一个蛋白质进行了详细的表征。拟南芥叶绿体铜转运蛋白COPT5属于真核生物Ctr家族，催化铜离子从液泡输出，并负责铜在器官间的分配。在第二个资助期内，我们将扩大对其他非生物因素如干旱、盐、热和渗透胁迫诱导的细胞膜蛋白质组变化的比较分析。与此同时，我们将研究这些应激因子如何改变张力体蛋白的磷酸化，以了解这种翻译后蛋白修饰与张力体转运机制调节的相关性。最后，我们将研究单个张力质体蛋白与植物胁迫反应的相关性，并努力阐明迄今为止未知的张力质体蛋白的生化和生理功能。

Proteom analyses represent a formidable tool to identify vacuolar proteins and to enlighten their relevance in stress response. During the past period of the research grant we have mainly focused on establishing a platform to isolate highly pure vacuoles applicable for mass spectrometry based proteomics. We isolated vacuoles from cold incubated and control plants to investigate changes in the tonoplast proteome during the process of cold acclimation. Thereby several changes in the abundance of tonoplast proteins and alterations in protein phosphorylation were identified providing first insights into the physiological relevance of the vacuole on cold acclimation. By these proteome analyses we identified several unknown proteins residing in the tonoplast and characterized one of these in detail. The tonoplast copper transporter COPT5 belongs to the eukaryotic Ctr family, catalyses the export of copper ions from the vacuole and is responsible for the inter organ allocation of copper in Arabidopsis. In the proposed second funding period we will expand the comparative analysis of changes in the proteome of the tonoplast induced by other abiotic factors like drought, salt, heat and osmotic stress. In parallel we will investigate how such stress factors alter the phosphorylation of tonoplast proteins to understand the relevance of this post-translational protein modification on the regulation of tonoplast transport mechanisms. Finally, we will investigate the relevance of individual tonoplast proteins for the plants stress response and we will effort to clarify the biochemical and physiological function of so far unknown tonoplast proteins.