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双伙伴分泌 (TPS-) 途径专门用于毒力因子（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同源 TpsB 蛋白形成的 β 桶孔输出。一个相关的转运系统是自转运蛋白，其中分泌的蛋白质在 C 端融合到介导跨外膜易位的 β 桶结构域。最近，我们开发了一种新型体外系统，用于研究 TpsA 蛋白跨 TpsB 孔的易位。我们现在想要使用该系统来识别参与陪伴并将 TpsA 蛋白靶向 TpsB 孔的周质蛋白。我们将使用位点特异性光交联与 TpsB 孔中停滞的 TpsA 蛋白易位中间体的生成相结合，以确定 TpsA 和 TpsB 之间的相互作用位点、跨 TpsB 的实际易位路径以及 TpsA 穿入 TpsB 的方向。通过使用 TpsA 蛋白的失活突变，我们想要检查其易位后折叠是否提供了运输的驱动力和单向性。我们将进一步利用无细胞系统来研究相关的自转运途径。

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