Metabolic coupling of plastidic and cytosolic pools of low-molecular weight thiols via members of the CRT-like transporter (CLT) family

谷胱甘肽是几乎所有生物体中最丰富的低分子量硫醇，在细胞氧化还原稳态和环境信号传导中具有重要功能。在拟南芥中，谷胱甘肽的两步生物合成途径在质体和细胞质之间进行区室化，其中第一种酶谷氨酸-半胱氨酸连接酶（GSH 1）仅位于质体中，而第二种酶谷胱甘肽合成酶（GSH 2）在两个区室中表达。耦合的两个谷胱甘肽池介导的最近确定的氯喹抗性转运蛋白样转运蛋白（CLTs），这是第一个功能特征的植物药物/代谢产物输出家族的成员。在这个项目中，蛋白质结构和所有三个拟南芥CLTs的运输特性的表征将在体内以及在体外异源表达系统和重组脂质体进行研究。CLTs在质体和胞质溶胶之间双向转运的体内功能将通过突变体背景中谷胱甘肽生物合成途径的隔室特异性重建进行分析。GSH 1在胞质溶胶中的错误表达也将提供有关GSH 1活性调节的新信息。在体内成像的谷胱甘肽氧化还原电位与氧化还原敏感的绿色荧光蛋白探针将进一步解决动态的谷胱甘肽氧化还原缓冲液下暴露于非生物和生物胁迫在亚细胞水平，并有助于阐明谷胱甘肽在激活植物免疫反应的作用。

Glutathione is the most abundant low molecular weight thiol in virtually all living organisms and has essential functions in cellular redox homeostasis and environmental signaling. In Arabidopsis thaliana, the two step biosynthetic pathway for glutathione is compartmentalized between plastids and cytosol with the first enzyme, glutamate-cysteine ligase (GSH1), being localized exclusively in the plastids and the second enzyme, glutathione synthase (GSH2), being expressed in both compartments. Coupling of the two glutathione pools is mediated by the recently identified chloroquine-resistance transporter like transporters (CLTs), which are the first functionally characterized members of the plant drug/metabolite exporter family. In this project, the protein structure and the characterization of the transport properties of all three Arabidopsis CLTs will be investigated in vivo as well as in vitro in heterologous expression systems and reconstituted liposomes. The in vivo function of CLTs for bidirectional transport between plastids and cytosol will be analyzed through compartment-specific reconstitution of the glutathione biosynthetic pathway in a mutant background. Misexpression of GSH1 in the cytosol will also provide novel information about the regulation of GSH1 activity. In vivo imaging of the glutathione redox potential with redoxsensitive GFP probes will further resolve the dynamics of the glutathione redox buffer under exposure to abiotic and biotic stress at subcellular level and help elucidating the role of glutathione in activating the immune response in plants.