Towards the membrane topology of the early translocation intermediate Ti-1 during Tat-dependent protein transport

双精氨酸转位(TAT)途径在叶绿体的类囊体膜以及细菌和古生菌的质膜上进行蛋白质转运，其独特之处在于它能将完全折叠的蛋白质跨离子密膜转运。它依赖于携带一对精氨酸残基的信号肽，可以分为四个连续的步骤：(1)前体蛋白与靶膜的非辅助结合，(2)与TAT受体的结合，(3)乘客蛋白的膜转位，以及(4)运输信号的蛋白水解性去除。最初的膜结合步骤导致早期转位中间产物Ti-1的形成，尽管目前还缺乏确切的数据，但人们认为这一步骤会导致TAT底物插入到脂质双层中。因此，我们建议为嵌合模型衬底16/23详细确定Ti-1的膜拓扑结构。利用一系列分子、生化和细胞生物学方法，我们想要检查16/23嵌合体是否在膜内形成完整的环拓扑，或者它是否部分地插入膜，或者甚至只是牢固地附着在脂质双层上，以防止完全的蛋白质降解。此外，我们建议确定TAT底物在其Ti-1构象中的低聚状态，以检查其与运输机制的相关性。

Protein transport by the twin-arginine translocation (Tat) pathway, which operates at the thylakoid membrane of chloroplasts and at the plasma membranes of bacteria and archaea, is unique with respect to its property to translocate fully folded proteins across ion-tight membranes. It depends on signal peptides carrying a twin pair of arginine residues and can be divided into four consecutive steps, (i) unassisted binding of the precursor protein to the target membrane, (ii) association with the Tat receptor, (iii) membrane translocation of the passenger protein, and (iv) proteolytic removal of the transport signal. The initial membrane binding step, which leads to the formation of the early translocation intermediate Ti-1, is assumed to result in the insertion of the Tat substrate into the lipid bilayer, although firm data are lacking yet. Therefore, we propose to determine in detail the membrane topology of Ti-1 for the chimeric model substrate 16/23. Employing a set of molecular, biochemical and cell biology approaches we want to examine if the 16/23 chimera develops an integral loop topology within the membrane or if it is partially membrane inserted or even only firmly attached to the lipid bilayer in a conformation that prevents complete proteolytic degradation. Furthermore, we propose to determine the oligomeric state of the Tat substrate in its Ti-1 conformation to examine its relevance for the transport mechanism.