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定位于特定的膜结合间隔来建立细胞组织。大多数靶向过程涉及到膜双层内或跨膜双层的转位。这是通过包含在每个前体蛋白的初级序列中的地址信号和结合信号并促进跨膜运输的细胞机制来实现的。许多蛋白质运输系统使用ATP或GTP马达来移动未折叠的蛋白质通过跨膜通道。然而，最近发现的一种名为Tat的系统，即双精氨酸转移，是不寻常的，因为它通过密封的膜运输完全折叠的蛋白质。蛋白质运输在所有细胞中都是一个基本的重要过程，许多致命的人类疾病都是由运输错误造成的。TAT系统在人类传染性疾病中发挥着特殊的作用，因为某些病原体依赖TAT获得毒力。我们的长期目标是利用叶绿体TAT系统(称为cpTat)作为实验模型来了解TAT蛋白的运输机制。CpTat是目前对TAT机制进行生化剖析的最佳系统。CpTat通过两个亚复合体可逆结合形成瞬时转位酶，即促进转运的酶复合体的循环过程而起作用。一个受体复合体与双胞胎精氨酸信号结合，似乎是蛋白质传导成分tha4的前体。尽管一些模型使用了适合形状的通道来运输，但我们的数据显示了一些完全不同的东西。我们发现，tha4在转位酶上经历了一个与通道组织不一致的主要构象变化。转位途径的间接校准意味着一个高度动态和瞬时的结构。在这里，我们提出了一种生化方法来确定转位酶在转位前和转位过程中的特性，这种方法可以稳定组装的转位酶。成分的身份(S)前体穿过膜并可能排列在路径上时的接触将通过专门设计的捕捉这种相互作用的前体来确定。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，甚至可能实现对纳米颗粒跨生物膜传输的现实工程和明确的控制。蛋白质运输在所有细胞中都是一个基本的重要过程，许多致命的人类疾病都是由运输错误造成的。TAT蛋白运输系统在健康中发挥着特殊的作用，因为某些病原体利用TAT来产生毒力。例子包括铜绿假单胞菌(Voulhoux等人，2001年)、结核分枝杆菌(McDonough等人，2005年)、大肠杆菌0157：H7(Pradel等人，2003年)、嗜肺军团菌(军团病)(De Buck等人，2005年)和幽门螺杆菌(Olson和Maier，2002年)。针对病原体TAT系统的治疗剂可能副作用较少，因为动物中没有TAT系统。公共卫生相关性：我们的研究可能导致此类试剂的出现，也可能导致设计纳米粒子跨生物膜传输的策略。

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.