Characterisation of the assembled state of the Tat protein transport system

所有的细菌，无论它们是否“友好”，都有一个共同点。为了殖民他们的利基市场，他们需要与外部世界交流。它们通过分泌蛋白质分子来实现这一点，这些蛋白质分子允许它们首先检测并操纵环境。细菌被一个或多个膜和坚硬的壁所包围，它们共同形成了一个保护性屏障。将蛋白质分泌到环境中需要这些分子能够通过膜屏障。为了实现这一点，细菌在细胞膜上有转运体，允许蛋白质向外传递。如果我们想要控制这个过程来预防疾病，或者设计微生物来净化有毒环境，了解这些蛋白质转运体的工作方式是至关重要的。我们研究了一种名为TAT系统的蛋白质转运体，它在几乎所有细菌和植物叶绿体中都是保守的，我们使用大肠杆菌作为一个方便的模型系统来研究这些过程。TAT系统在许多不同细菌的生理中起着非常重要的作用，对植物的光合作用也是必不可少的。蛋白质是由长的、线性的氨基酸链组成的，这些氨基酸在形成后折叠起来。蛋白质只有在折叠成最终的三维结构后才具有功能。分泌的蛋白质在细菌细胞外具有功能。TAT系统是不同寻常的，因为与大多数其他蛋白质转运体不同，它只在蛋白质折叠后才进行转运体。因为不同的折叠蛋白质有不同的大小，这意味着TAT系统必须能够形成通道，能够容纳它运输的不同直径的折叠蛋白质。这是如何实现的？TAT系统注定要分泌的蛋白质有一个特殊的标志性氨基酸序列，在它们开始时被称为“双胞胎精氨酸信号”。这个信号将蛋白质定位于嵌入在膜中的TAT机器，并促进其分泌。TAT机器本身由三个组件组成--TATA、TATB和TATC。TatB和TatC组分相互形成1：1的复合体，该复合体通过与双胞胎精氨酸信号相互作用，负责识别TAT系统靶向分泌的不同蛋白质。在信号被TatBC结合后，这会触发TATA组件组装成环状结构，从而允许蛋白质的运输。在蛋白质被运输后，塔塔环会解体，准备进行另一轮分泌。我们已经能够分离出允许TAT系统运输没有双胞胎精氨酸信号的蛋白质的突变体。我们想了解这些突变的TAT系统是如何工作的--它们如何识别要运输的蛋白质？这将有助于我们理解双精氨酸信号是如何激活TAT系统的。从长远来看，这些突变的TAT系统可能会有用，因为它们可能能够分泌各种不同的蛋白质，使我们能够将它们开发成细胞工厂，以生产和分泌重要的工业蛋白质。

All bacteria, whether they are 'friendly' or otherwise, have one thing in common. In order to colonize their niches they need to communicate with the outside world. They achieve this by secreting protein molecules that allow them firstly to detect and then manipulate their environment. Bacteria are surrounded by one or more membranes and a rigid wall, which together form a protective barrier. Secreting proteins into the environment requires that these molecules are able to pass through the membrane barrier. In order to achieve this, bacteria have transporters located in the membrane that allow the passage of proteins to the outside. Understanding how these protein transporters work is critical if we wish to control this process to prevent disease, or engineer microbes to decontaminate toxic environments.We study a protein transporter called the Tat system, that is conserved in almost all bacteria and in plant chloroplasts, and we use E. coli as a convenient model system in which to study these processes. The Tat system plays a very important role in the physiology of many different bacteria and it is essential for photosynthesis in plants.Proteins are made up of long, linear chains of amino acids which fold up after they are made. Proteins are only functional once they have folded into their final 3-dimensional structure. Proteins that are secreted are functional outside the bacterial cell. The Tat system is unusual because unlike most other protein transporters it only transports proteins after they have already folded. Because different folded proteins have different sizes, this means that the Tat system must be able to form channels that can accommodate the different diameters of the folded proteins that it transports. How is this achieved?Proteins that are destined to be secreted by the Tat system have a special signature sequence of amino acids, termed a 'twin arginine signal' at their start. This signal targets the protein to the Tat machinery that is embedded in the membrane, and facilitates its secretion. The Tat machinery itself is made up of three components - TatA, TatB and TatC. The TatB and TatC components form a 1:1 complex with each other and this complex is responsible for recognizing each of the different proteins that are targeted for secretion by Tat system, by interacting with the twin arginine signal. After the signal has been bound by TatBC this triggers the TatA component to assemble into a ring-like structure, which can then allow transport of the protein. After the protein has been transported the TatA ring disassembles ready for another round of secretion. We have been able to isolate mutants that allow the Tat system to transport proteins that have no twin arginine signal. We would like to understand how these mutated Tat systems work - how do they identify proteins to transport? This will help us to understand how the twin arginine signal is able to activate the Tat system. In the long run these mutated Tat systems have the potential to be useful because they may be able to secrete a wide range of different proteins, allowing us to develop them into cell factories to produce and secrete important industrial proteins.