Genetic Analysis of the Escherichia coli Tat Pathway

描述（由申请人提供）：双精氨酸转运蛋白（达特）是最近发现的细菌和植物中的蛋白质分泌途径。本提案的总体目标是利用遗传和生物化学手段阐明达特途径的蛋白质分泌机制。本申请的一个独特之处是PI开发了新型高通量筛选方法，极大地帮助了达特途径的遗传解剖。 所提出的研究集中在达特输出的一些关键的早期步骤的解剖：具体目标1将集中在前导肽的结合和易位子复合物的组装：1.1达特前导肽的特征的分析，所述特征介导它们被达特装置识别，以及达特前导肽的子集避免错误路由到Sec的机制。1.2负责前导肽内达特共有基序结合的TatC区的遗传鉴定。1.3利用一个有缺陷的前导肽及其识别的TatC抑制子，该抑制子仅特异性识别该前导肽（通过1.2中的研究分离），用于达特蛋白寡聚化变化的生化分析和导致形成转位子复合物的事件。 在具体目标2下的研究中，我们将检查达特的折叠质量控制特征。我们将努力：2.1分离使途径的折叠质量控制特征失效的达特突变，以允许未折叠多肽的输出。2.2检查TatB的作用，特别是其胞质结构域，我们的初步研究显示其具有伴侣活性，因此可能负责未折叠的成熟多肽的结合，并详细研究底物蛋白的折叠动力学和达特输出能力之间的关系。 总的来说，本申请中提出的研究将导致我们对达特途径的理解的重大进展。修订后的申请可能会产生显着的和完全独特的见解达特途径，除了他们的机械意义，将是相当大的医疗效益，鉴于达特出口在细菌致病性的重要性。

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.