Targeting and Assembly of thylakoid membrane proteins

描述(申请人提供)：在真核细胞中，新合成的蛋白质被特异性地从它们的合成部位转移到它们的功能部位。在大多数情况下，这涉及到向至少一个膜内或跨膜转运。一般来说，这些运输步骤涉及靶向每个蛋白质的初级序列和细胞机制中包含的元件，以识别和促进前体蛋白质的易位。最近的研究已经在叶绿体的类囊体膜以及细菌和古生菌的细胞质膜中发现了被称为TAT的同源蛋白质转运系统。TAT系统仅使用跨膜质子梯度作为能量来源，就可以独特地运输折叠蛋白质。他们只用机器的三个膜组件就能做到这一点，而且不会破坏膜的渗透性。TAT系统在植物和一些原核生物中是必不可少的。重要的是，至少有一种人类病原体使用TAT系统将毒力因子传递给宿主。由于哺乳动物基因组中似乎缺少TAT成分，TAT系统是新型抗菌化合物的潜在靶点。我们的长期目标是确定TAT系统转运蛋白质的机制。我们最近的研究描述了这一过程的步骤，并确定了三个已知成分(cpTatC、Hcf106和tha4)中的哪一个参与每个步骤。具体地说，前体与由cpTatC和Hcf106组成的700 kDa受体复合体结合。前体结合和质子梯度触发TH4与受体复合体的组装。然后前体跨膜运输，转位复合体解离。在这里，我们提出了一系列关于易位机制的生化研究。具体地说，纯化和交联研究将表征受体复合体的组成、原位大小、信号肽结合能力和结合部位，以及它在信号结合时所经历的变化。一项新开发的生化互补分析将研究tha4寡聚在不同大小的折叠前体易位中的作用。直接和间接方法将测试转位酶结构和操作的两种替代模型。这里提出的工作将增加对蜂窝机器机制的基本知识。它还可能为解决人类微生物疾病提供新的战略。

DESCRIPTION (provided by applicant): In eukaryotic cells, newly made proteins are specifically transferred from their sites of synthesis to their sites of function. In most cases, this involves transport into or across at least one membrane. In general, these transport steps involve targeting elements contained within each protein's primary sequence and cellular machinery to recognize and facilitate translocation of the precursor protein. Recent studies have identified homologous protein translocation systems called "Tat" in the thylakoid membranes of chloroplasts and the cytoplasmic membranes of bacteria and archaea. Tat systems uniquely transport folded proteins using only the transmembrane proton gradient as an energy source. They do this with only three membrane components of machinery and without breaching the permeability of the membrane. The Tat system is essential in plants and some prokaryotes. Importantly, the Tat system is used by at least one human pathogen to deliver virulence factors to its hosts. As Tat components seem absent from mammalian genomes, the Tat system represents a potential target for novel antimicrobial compounds. Our long range goal is to determine the mechanism by which Tat systems translocate proteins. Our recent studies have described steps of the process and identified which of the three known components (cpTatC, Hcf106, and Tha4) participate in each step. Specifically, precursors bind to a 700 kDa receptor complex consisting cpTatC and Hcf106. Precursor binding and the proton gradient trigger assembly of Tha4 to the receptor complex. The precursor is then transported across the membrane and the translocation complex dissociates. Here we propose a series of biochemical studies into the mechanism of translocation. Specifically, purification and crosslinking studies will characterize the receptor complex composition, in situ size, signal peptide binding capabilities and binding site, and changes that it undergoes upon signal binding. A newly developed biochemical complementation assay will investigate the role of Tha4 oligomerization in translocation of folded precursors of varied size. Direct and indirect approaches will test two alternative models for the structure and operation of the translocase. The work proposed here will increase basic knowledge of the mechanisms of cellular machines. It may additionally provide for new strategies to address human microbial diseases.