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

双精氨酸转运（达特）途径是独特的，其性质，以完全折叠构象跨离子密封膜转运蛋白质。它由膜电位驱动，膜电位可以是Δ pH和/或Δ psi。在叶绿体和革兰氏阴性细菌中，达特移位酶由被认为构成膜受体的完整亚基TatB和TatC以及TatA组成，TatA是一种双位点膜蛋白，以一种尚不清楚的方式负责实际的膜移位步骤。另外，在叶绿体基质中也发现了TatA，并且最近表明，在E. coli中，可以在功能上替代固有的类囊体TatA活性。该方法允许我们精确地定量在标准条件下模型达特底物16/23的类囊体转运所需的TatA的量（Hauer等人，2013年）。在这里，我们建议扩展TatA的需求在类囊体运输的一组前体蛋白，彼此不同的分子量，尺寸和/或折叠状态的乘客多肽，以及关于信号肽存在的定量分析。运输实验将在不同的生理条件下进行，例如温度和/或光照条件，以考虑实验环境对TatA要求的任何潜在影响。最后，研究了TatA非结构化C-末端结构域在转运过程中的作用。为此目的，在大肠杆菌中异源过表达后将纯化不同的TatA衍生物。coli中，并在这种定量重建实验中进行分析，即具有融合到其C-末端的EGFP的嵌合TatA蛋白以及缺乏部分C-末端结构域的TatA截短衍生物。

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.