Substrate-translocase interactions during two-partner secretion in Gram-negative bacteria

革兰氏阴性菌两伙伴分泌过程中底物-转位酶的相互作用

• 批准号: 61723904
• 负责人: Professor Dr. Matthias Müller
• 金额: --
• 依托单位: Institut für Biochemie und Molekularbiologie
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/61723904?language=en
• 关键词: Substrate; translocase; interactions; during; two

The two-partner secretion (TPS-) pathway found in pathogenic Gram-negative bacteria is dedicated to the secretion of virulence factors (termed TpsA-proteins). TpsA-proteins are translocated via the Sec translocon to the periplasm, from where they are exported through a β-barrel pore that is formed by the cognate TpsB protein in the outer membrane. A related transport system is that of autotransporters, in which the secreted protein is C-terminally fused to a β-barrel domain that mediates translocation across the outer membrane. Recently we have developed a novel in vitro system for the study of translocation of a TpsA protein across its TpsB-pore. We now want to use this system to identify periplasmic proteins involved in chaperoning and targeting a TpsA protein to the TpsB-pore. We will use site-specific photo-crosslinking in combination with the generation of translocation intermediates of a TpsA-protein stalled in the TpsB-pore, to determine the interacting sites between TpsA and TpsB, the actual translocation path across TpsB, and the orientation by which TpsA is threaded into TpsB. By use of inactivating mutations of the TpsA-protein we want to examine if its post-translocational folding provides the driving force and unidirectionality of transport. We will further exploit the cell-free system to study the related autotransporter pathway.

在致病性革兰氏阴性菌中发现的两个伙伴分泌(TPS-)途径致力于毒力因子(称为TPSA-蛋白)的分泌。TPSA-蛋白通过SEC易位子转运到周质，从那里它们通过外膜中的同源tPSB蛋白形成的β桶孔输出。一个相关的转运系统是自体转运蛋白，其中分泌的蛋白在C端融合到一个β桶结构域，该结构域介导外膜的转运。最近，我们开发了一种新的体外系统来研究TPSA蛋白在其TPSB孔中的易位。我们现在希望使用这个系统来识别参与伴侣作用的周质蛋白，并将TPSA蛋白定位到TPSB孔。我们将使用定点光交联法结合产生停滞在TPSB孔中的TPSA蛋白的易位中间体，来确定TPSA和TPSB之间的相互作用部位，实际的穿过TPSB的易位路径，以及TPSA被穿入TPSB的方向。通过利用TPSA蛋白的失活突变，我们想要检查它的移位后折叠是否提供了运输的驱动力和单向性。我们将进一步开发无细胞系统来研究相关的自体转运蛋白途径。

项目成果

Mapping Precursor-binding Site on TatC Subunit of Twin Arginine-specific Protein Translocase by Site-specific Photo Cross-linking*

• DOI: 10.1074/jbc.m112.343798
• 发表时间: 2012-02
• 期刊: The Journal of Biological Chemistry
• 作者: Stefan Zoufaly;J. Fröbel;Patrick H Rose;T. Flecken;C. Maurer;Michael Moser;Matthias Müller

Versatile in vitro system to study translocation and functional integration of bacterial outer membrane proteins

• DOI: 10.1038/ncomms6396
• 发表时间: 2014-11
• 期刊: Nature Communications
• 影响因子: 16.6
• 作者: Derrick Norell;A. Heuck;T. Tran‐Thi;H. Götzke;F. Jacob-Dubuisson;T. Clausen;D. Daley;V. Braun;Matthias Müller;Enguo Fan