Targeting and Assembly of Thylakoid Membrane Proteins

描述（由申请人提供）：高等生物通过将蛋白质靶向特定的膜结合区室来建立细胞组织。大多数靶向过程涉及易位进入或穿过膜双层。这是通过寻址包含在每个前体蛋白的一级序列和细胞机器内的信号来实现的，以结合信号并促进跨膜转运。许多蛋白质转运系统使用ATP或GTP马达通过跨膜通道移动未折叠的蛋白质。然而，最近发现的一种称为达特的系统，即双精氨酸易位系统，是不寻常的，因为它可以将完全折叠的蛋白质运输穿过密封的膜。蛋白质运输是所有细胞中的一个基本重要过程，许多致命的人类疾病都是由运输错误引起的。达特系统在传染性人类疾病中发挥特定作用，因为某些病原体依赖于达特的毒力。我们的长期目标是利用叶绿体达特系统（简称cp达特系统）作为实验模型来了解达特蛋白的转运机制。cp达特是目前对达特机制进行生化解剖的最佳系统。cpTat通过一个循环过程进行操作，其中两个亚复合物可逆地结合以形成瞬时易位酶，即促进转运的酶复合物。受体复合物结合双精氨酸信号，并似乎呈现蛋白质传导组分Tha4的前体。虽然有些模型调用形状拟合通道的传输，我们的数据表明完全不同的东西。我们发现，Tha4经历了一个重大的构象变化的转位酶是不一致的通道组织。间接校准的易位途径意味着一个高度动态和瞬态的结构。在这里，我们提出了一种生物化学的方法来确定易位酶的特性之前和易位过程中的方法，可以稳定组装的易位酶。当前体穿过膜并可能沿着路径时与前体接触的成分的身份将通过旨在捕获此类相互作用的专门前体来确定。这些目标的成功实现将解决一个长期存在的科学难题，可能导致高度特异性的治疗剂，甚至可能允许现实的工程和确定的控制纳米尺寸的颗粒通过生物膜的运输。蛋白质运输是所有细胞中的一个基本重要过程，许多致命的人类疾病都是由运输错误引起的。达特蛋白转运系统在健康中发挥特定作用，因为某些病原体利用达特进行毒力。实例包括铜绿假单胞菌（Voulhoux等人，2001）、结核分枝杆菌（麦克多诺等人，2005），E.大肠杆菌0157：H7（Pradel等人，2003）、嗜肺军团菌（Legionella pneumophila）（军团病）（De Buck等人，2005）和幽门螺杆菌（Olson和Maier，2002）。靶向病原体的达特系统的治疗剂可能具有较少的副作用，因为动物不存在达特系统。公共卫生相关性：我们的研究可能会导致这样的代理商，也可能导致工程纳米粒子跨生物膜运输的策略。

DESCRIPTION (provided by applicant): Higher organisms establish cellular organization by targeting proteins to specific membrane bounded compartments. Most targeting processes involve translocation into or across a membrane bilayer. This is achieved by address signals contained within each precursor protein's primary sequence and cellular machinery to bind the signals and facilitate transmembrane transport. Many protein transport systems use ATP or GTP motors to move unfolded proteins through transmembrane channels. However, a recently discovered system called Tat, for Twin arginine translocation, is unusual because it transports fully folded proteins across sealed membranes. Protein transport is a fundamentally important process in all cells and numerous fatal human diseases result from trafficking errors. Tat systems play specific roles in infectious human diseases because certain pathogens rely on Tat for virulence. Our long range goal is to understand the mechanism of Tat protein transport using the chloroplast Tat system (called cpTat) as an experimental model. cpTat is currently the best system for biochemical dissection of Tat mechanism. cpTat operates by a cyclical process in which two subcomplexes reversibly associate to form a transient translocase, i.e. the enzyme complex that facilitates transport. A receptor complex binds the twin arginine signal and appears to present the precursor to the protein conducting component, Tha4. Although some models invoke form-fitting channels for transport, our data suggest something quite different. We found that Tha4 undergoes a major conformational change in the translocase that is not consistent with a channel organization. Indirect calibration of the translocation pathway implies a highly dynamic and transient structure. Here we propose a biochemical approach to determine characteristics of the translocase both before and during translocation with a method that can stabilize an assembled translocase. The identity of component (s) that contact of the precursor as it goes across the membrane and presumably line the pathway will be determined with specialized precursors designed to capture such interactions. The successful accomplishment of these goals will solve a longstanding scientific puzzle, may lead to highly specific therapeutic agents, and may even allow realistic engineering and defined control of nanometer sized particle transport across biological membranes. Protein transport is a fundamentally important process in all cells and numerous fatal human diseases result from trafficking errors. Tat protein transport systems play specific roles in health because certain pathogens employ Tat for virulence. Examples include Pseudomonas aeruginosa (Voulhoux et al., 2001), Mycobacterium tuberculosis (McDonough et al., 2005), E. coli 0157:H7 (Pradel et al., 2003), Legionella pneumophila (Legionnaires disease) (De Buck et al., 2005), and Helicobacter pylori (Olson and Maier, 2002). Therapeutic agents that target the Tat system of pathogens are likely to have fewer side effects because Tat systems are absent from animals. Public Health Relevance: Our studies could lead to such agents and may also lead to strategies for engineering nano-particle transport across biological membranes.