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进入或穿过膜双分子层的易位。这是通过定位每个前体蛋白初级序列中的信号和细胞机制来实现的，以结合信号并促进跨膜运输。许多蛋白质运输系统使用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Tat（双精氨酸易位）的系统是不寻常的，因为它通过密封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至关重要的过程，许多致命的人类疾病都是由于运输错误造成的。Tat系统在人类传染性疾病中起着特殊的作用，因为某些病原体依靠Tat产生毒力。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蛋白运输的机制。cpTat是目前对Tat机制进行生化解剖的最佳体系。cpTat通过一个循环过程起作用，其中两个亚复合物可逆地结合形成瞬时转位酶，即促进运输的酶复合物。受体复合体结合双精氨酸信号，似乎是蛋白质传导成分Tha4的前体。虽然有些模型调用了适合运输的通道，但我们的数据表明了一些完全不同的东西。我们发现，Tha4在转位酶中经历了与通道组织不一致的主要构象变化。间接校准的易位路径意味着一个高度动态和瞬态结构。在这里，我们提出了一种生化方法来确定易位酶在易位之前和过程中的特征，这种方法可以稳定组装的易位酶。当前体穿过膜并沿着途径排列时，与前体接触的成分的身份将由专门设计用于捕获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，可能会导致高度特异性的治疗药物，甚至可能允许现实的工程和定义控制纳米级颗粒在生物膜上的运输。蛋白质运输在所有细胞中都是一个至关重要的过程，许多致命的人类疾病都是由于运输错误造成的。Tat蛋白运输系统在健康中起着特殊的作用，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更少，因为动物体内没有Tat系统。公共卫生相关性：我们的研究可能会导致这样的药物，也可能导致工程纳米颗粒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.