Demand of TatA during Tat-dependent protein transport across the thylakoid membrane

双精氨酸易位（Tat）途径是独特的，就其性质而言，以一个完全折叠的构象跨离子紧密膜易位蛋白质。它是由膜电位驱动的，可以是pH和/或psi。在叶绿体和革兰氏阴性菌中，Tat转位酶由完整的亚基TatB和TatC组成，它们被认为构成了膜受体，TatA是一种双位膜蛋白，以一种未知的方式负责实际的膜易位步骤。此外，TatA也存在于叶绿体基质中，最近的研究表明，这种在大肠杆菌中异种表达后纯化的可溶性TatA可以在功能上取代固有的类囊体TatA活性。这种方法使我们能够准确量化标准条件下模型Tat底物16/23类囊体运输所需的TatA量（Hauer等人，2013）。在这里，我们建议将类囊体运输过程中TatA需求的定量分析扩展到一组前体蛋白，这些前体蛋白在分子质量、尺寸和/或乘客多肽的折叠状态以及存在的信号肽方面彼此不同。运输实验将在不同的生理条件下进行，例如不同的温度和/或光照条件，以便考虑到实验环境对TatA要求的任何潜在影响。最后，研究TatA的非结构化c端结构域在转运过程中的作用。为此，将在大肠杆菌中外源过表达后纯化不同的TatA衍生物，并在这种定量重构实验中进行分析，即将EGFP融合到其c端的嵌合TatA蛋白以及缺少部分c端结构域的TatA截断衍生物。

The twin-arginine translocation (Tat) pathway is unique with respect to its property to translocate proteins in a fully folded conformation across ion-tight membranes. It is driven by the membrane potential which can be delta pH and/or delta psi. In chloroplasts and Gram-negative bacteria, Tat translocase consists of the integral subunits TatB and TatC, which are assumed to constitute the membrane receptor, and TatA, a bitopic membrane protein being responsible in a yet unknown manner for the actual membrane translocation step. TatA is additionally found also in the chloroplast stroma and it was recently shown that such soluble TatA, which was purified after heterologous expression in E. coli, can functionally replace the intrinsic thylakoidal TatA activity. This approach allowed us to exactly quantify the amount of TatA that is required for thylakoid transport of the model Tat substrate 16/23 under standard conditions (Hauer et al., 2013). Here we propose to extend the quantitative analysis of TatA demand during thylakoid transport to a set of precursor proteins that differ from each other in terms of molecular mass, dimensions, and/or folding status of the passenger polypeptide as well as with regard to the signal peptide present. The transport experiments will be performed under different physiological conditions, varying for example temperature and/or light regime, in order to take into account any potential influence of the experimental circumstances on TatA requirement. And finally, the role of the unstructured C-terminal domain of TatA in the transport process is to be studied. For this purpose, different TatA derivatives will be purified after heterologous overexpression in E. coli and analysed in such quantitative reconstitution experiments, namely a chimeric TatA protein having EGFP fused to its C-terminus as well as TatA truncation derivatives lacking parts of the C-terminal domain.