Targeting and Assembly of thylakoid membrane proteins

描述（由申请人提供）：在真核细胞中，新产生的蛋白质从其合成位点特异性转移到其功能位点。在大多数情况下，这涉及转运进入或穿过至少一个膜。一般来说，这些转运步骤涉及包含在每种蛋白质的一级序列和细胞机器内的靶向元件，以识别和促进前体蛋白的易位。最近的研究发现，在叶绿体的类囊体膜和细菌、古细菌的细胞质膜中存在一种称为“达特”的同源蛋白质易位系统。达特系统仅使用跨膜质子梯度作为能量来源来独特地运输折叠蛋白质。他们这样做，只有三个膜组件的机械和不破坏渗透性的膜。达特系统在植物和某些原核生物中是必需的。重要的是，达特系统被至少一种人类病原体用于将毒力因子递送至其宿主。由于哺乳动物基因组中似乎不存在达特组分，因此达特系统代表了新型抗微生物化合物的潜在靶标。我们的长期目标是确定达特系统转运蛋白的机制。我们最近的研究已经描述了该过程的步骤，并确定了三种已知组分（cpTatC，Hcf106和Tha4）中的哪一种参与了每个步骤。具体地，前体结合到由cpTatC和Hcf106组成的700 kDa受体复合物。前体结合和质子梯度触发组装Tha4的受体复合物。然后前体被转运穿过膜，并且易位复合物解离。在这里，我们提出了一系列的生化研究易位的机制。具体而言，纯化和交联研究将表征受体复合物的组成、原位大小、信号肽结合能力和结合位点，以及其在信号结合时所经历的变化。一种新开发的生化互补试验将研究Tha4寡聚化在不同大小的折叠前体易位中的作用。直接和间接的方法将测试两种替代模型的结构和运作的易位酶。这里提出的工作将增加细胞机器的机制的基础知识。它还可以提供新的策略来解决人类微生物疾病。

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.