Characterisation of the assembled state of the Tat protein transport system

所有的细菌，不管它们是“友好的”还是其他的，都有一个共同点。为了开拓他们的利基市场，他们需要与外界沟通。它们通过分泌蛋白质分子来实现这一点，这些蛋白质分子使它们能够首先检测并操纵它们的环境。细菌被一个或多个膜和刚性壁包围，它们共同形成保护屏障。将蛋白质分泌到环境中需要这些分子能够穿过膜屏障。为了实现这一点，细菌具有位于膜中的转运蛋白，允许蛋白质通过到外部。如果我们希望控制这一过程以预防疾病，或设计微生物以净化有毒环境，那么了解这些蛋白质转运蛋白的工作方式至关重要。我们研究了一种称为达特系统的蛋白质转运蛋白，该系统在几乎所有细菌和植物叶绿体中都是保守的。大肠杆菌作为一个方便的模型系统，在其中研究这些过程。达特系统在许多不同细菌的生理学中起着非常重要的作用，它是植物光合作用所必需的。蛋白质是由长的线性氨基酸链组成的，它们在合成后折叠起来。蛋白质只有在折叠成最终的三维结构后才有功能。分泌的蛋白质在细菌细胞外起作用。达特系统是不寻常的，因为不像大多数其他蛋白质转运蛋白，它只运输蛋白质后，他们已经折叠。由于不同的折叠蛋白质具有不同的大小，这意味着达特系统必须能够形成能够容纳其运输的折叠蛋白质的不同直径的通道。这是如何实现的？注定由达特系统分泌的蛋白质具有特殊的氨基酸特征序列，在其起始处称为“双精氨酸信号”。该信号将蛋白质靶向嵌入膜中的达特机制，并促进其分泌。达特机器本身由三个部件组成- TatA、TatB和TatC。TatB和TatC组分彼此形成1：1复合物，并且该复合物负责通过与双精氨酸信号相互作用来识别被达特系统靶向分泌的每种不同蛋白质。在信号被TatBC结合后，这触发了TatA组分组装成环状结构，然后可以允许蛋白质的运输。蛋白质被运输后，TatA环分解，准备进行另一轮分泌。我们已经能够分离出允许达特系统转运没有双精氨酸信号的蛋白质的突变体。我们想了解这些突变的达特系统是如何工作的--它们是如何识别要运输的蛋白质的？这将有助于我们理解双精氨酸信号是如何激活达特系统的。从长远来看，这些突变的达特系统有潜力发挥作用，因为它们可能能够分泌各种不同的蛋白质，使我们能够将其发展成细胞工厂，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.