Genetic Analysis of the Escherichia coli Tat Pathway

描述(申请人提供)：双精氨酸转运体(Tat)是最近发现的细菌和植物中蛋白质分泌的途径。这项建议的总体目标是利用遗传和生化手段阐明TAT途径分泌蛋白质的机制。这一应用的一个独特特征是PI开发了新的高通量筛选方法，极大地帮助了TAT途径的遗传解剖。 建议的研究集中在对TAT输出的一些关键早期步骤的剖析：特定目标1将集中于前导肽的结合和转位复合体的组装：1.1分析TAT前导肽的特征，这些特征介导它们被TAT装置识别，以及TAT前导肽的子集避免错误地进入SEC的机制。1.2负责在前导肽中结合TAT共识基序的TatC区域的遗传鉴定。1.3开发有缺陷的前导肽及其已知的TatC抑制子，该前导肽仅专门识别该前导(通过1.2中的研究分离)，用于生化分析TAT蛋白齐聚的变化和导致转位复合体形成的事件。 在特定目标2下的研究中，我们将检查TAT的折叠质量控制功能。我们将寻求：2.1分离TAT突变，这些突变使该途径的折叠质量控制功能失效，从而允许未折叠多肽的出口。2.2研究TatB的作用，特别是其胞质结构域，我们的初步研究表明，TatB具有伴侣活性，因此可能与未折叠的成熟多肽的结合有关；2.3详细研究底物蛋白的折叠动力学与TAT输出能力之间的关系。 总体而言，本申请中提出的研究将使我们对TAT途径的理解有显著的进步。修订后的应用可能会对TAT途径产生重大和完全独特的见解，鉴于TAT出口在细菌致病性方面的重要性，除了它们的机制意义外，还将具有相当大的医学益处。

DESCRIPTION (provided by applicant): The Twin Arginine Transporter (Tat) is a recently discovered pathway for protein secretion in bacteria and plants. The overall objective of this proposal is to elucidate the mechanism of protein secretion by the Tat pathway using genetic and biochemical means. A unique feature of this application is the development by the PI of novel high throughput screening methodologies that have greatly aided the genetic dissection of the Tat pathway. The proposed studies are focused on the dissection of some of the key early steps in Tat export: Specific Aim 1 will focus on leader peptide binding and the assembly of the translocon complex: 1.1 The analysis of the features of Tat leader peptides that mediate their recognition by the Tat apparatus and also the mechanism by which a subset of Tat leader peptides avoid misrouting into Sec. 1.2 Genetic identification of the TatC region that is responsible for the binding of the Tat consensus motif within the leader peptide. 1.3 The exploitation of a defective leader peptide and its cognizant TatC suppressor that specifically recognizes only that leader (isolated though the studies in 1.2) for the biochemical analysis of changes in Tat protein oligomerization and the events that lead to the formation of the translocon complex. In studies under Specific Aim 2 we will examine the folding quality control feature of Tat. We will seek to: 2.1 Isolate Tat mutations that disable the folding quality control feature of the pathway to permit the export of unfolded polypeptides. 2.2 Examine the role of TatB, and specifically its cytoplasmic domain which our preliminary studies reveal to have chaperone activity and therefore may be responsible for the binding of unfolded mature polypeptides and 2.3 Investigate in detail, the relationship between folding kinetics of substrate proteins and Tat export competence. Overall, the studies proposed in this application will lead to a significant advance in our understanding of the Tat pathway. The revised application is likely to yield significant and completely unique insights into the Tat pathway that, apart from their mechanistic significance, will be of considerable medical benefit, given the established importance of Tat export in bacterial pathogenicity.