Functional reconstitution of thylakoidal Tat translocase (Twin arginine translocase) into lipid membranes

达特（twin arginine translocation）途径能够将完全折叠的蛋白质运输穿过离子不透膜，其在叶绿体的类囊体膜以及细菌和古细菌的细胞质膜上起作用。在叶绿体中，达特机制由三种膜蛋白TatA、TatB和TatC组成。TatB和TatC构成一组高分子量的异聚复合物，其中一些为携带Tat特异性信号肽的底物蛋白提供膜受体。TatA与这些受体复合物相互作用，并且是达特底物的实际膜易位所需的。目前讨论了三种模型，（a）具有不同或可变直径的易位孔，（B）TatA诱导的膜弱化，使得能够直接通过脂质双层易位，以及（c）TatA诱导TatBC复合物转运的催化或调节功能。尽管许多实验室进行了广泛的努力，但到目前为止还不可能用已建立的分析系统阐明转运机制。因此，我们建议从异源表达的纯化达特亚基开始在脂质体中重建功能性达特转运机制。脂质体不含类囊体膜的潜在干扰组分，因此应允许确定（i）达特亚基自组装成异聚体膜复合物的性质，（ii）确切的这些达特复合物内达特亚基的（和可能可变的）化学计量，（iii）脂质组合物对于复合物的组装和功能性的潜在作用，以及（iv）在运输过程的每个步骤中涉及任一子单元。从长远来看，这些实验也将为这种特殊的蛋白质运输机制的详细结构分析提供基础。

The Tat (twin arginine translocation) pathway, which is capable of transporting fully folded proteins across ion-tight membranes, is operating at the thylakoid membrane of chloroplasts as well as at the cytoplasmic membranes of bacteria and archaea. In chloroplasts, Tat machinery is composed of the three membrane proteins TatA, TatB, and TatC. TatB and TatC constitute a set of heteromeric complexes of high molecular weight, some of which provide the membrane receptors for substrate proteins carrying Tat-specific signal peptides. TatA interacts with these receptor complexes and is required for the actual membrane translocation of the Tat substrates. Three models are currently discussed, (a) translocation pores with different or variable diameter, (b) TatA-induced membrane weakening enabling translocation directly through the lipid bilayer, and (c) a catalytic or regulatory function of TatA inducing transport by the TatBC complexes. Despite extensive efforts from numerous labs it was not possible so far to clarify the transport mechanism with the established analytical systems. Therefore, we propose to reconstitute functional Tat translocation machinery in liposomes starting from heterologously expressed, purified Tat subunits. Liposomes are free from potentially interfering components of the thylakoid membrane and should thus allow to determine (i) the properties of the Tat subunits to self-assemble into heteromeric membrane complexes, (ii) the exact (and possibly variable) stoichiometry of Tat subunits within these Tat complexes, (iii) the potential role of lipid composition for the assembly and functionality of the complexes, and (iv) the involvement of either of the subunits in each step of the transport process. In the long term, these experiments will provide the basis also for detailed structural analyses of this exceptional protein transport machinery.