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

Tat 依赖性蛋白质转运过程中早期易位中间体 Ti-1 的膜拓扑

基本信息

批准号：
194404447
负责人：
Professor Dr. Ralf Bernd Klösgen
金额：
--
依托单位：
Abteilung Pflanzenphysiologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2011
资助国家：
德国
起止时间：
2010-12-31 至 2014-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/194404447?language=en
关键词：
Towards membrane topology early translocation

项目摘要

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.

双精氨酸转运（达特）途径是一种独特的蛋白质转运途径，它作用于叶绿体的类囊体膜和细菌、古细菌的质膜，能使完全折叠的蛋白质穿过不透离子的膜。它依赖于携带一对精氨酸残基的信号肽，并可分为四个连续步骤，（i）前体蛋白与靶膜的无辅助结合，（ii）与达特受体的结合，（iii）乘客蛋白的膜易位，和（iv）转运信号的蛋白水解去除。最初的膜结合步骤，这导致早期易位中间体Ti-1的形成，被认为是导致插入的达特基板到脂质双层，虽然还缺乏可靠的数据。因此，我们建议详细确定嵌合模型基质16/23的Ti-1的膜拓扑结构。采用一组分子，生物化学和细胞生物学方法，我们要检查16/23嵌合体是否在膜内形成完整的环拓扑结构，或者它是否部分插入膜，甚至仅牢固地附着在脂质双层上，以防止完全蛋白水解降解。此外，我们建议确定低聚状态的达特基板在其Ti-1构象，以检查其相关性的运输机制。