The integration of tail anchored membrane proteins by the twin-arginine translocase

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，这些蛋白质分子使它们能够首先检测并操纵它们的环境。细菌被一层或多层膜和一层坚硬的壁所包围，它们共同形成了一个保护屏障。将蛋白质分泌到环境中需要这些分子能够穿过膜屏障。为了达到这个目的，细菌的细胞膜上有转运蛋白，可以将蛋白质运送到细胞膜外。如果我们希望控制这一过程以预防疾病，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原理是至关重要的。我们研究了一种叫做Tat系统的蛋白质转运体，它在几乎所有的细菌和植物叶绿体中都是保守的，我们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，对植物的光合作用至关重要。蛋白质是由长而线性的氨基酸链组成的，这些氨基酸链在合成后会折叠起来。蛋白质只有在折叠成最终的三维结构后才能发挥作用。分泌的蛋白质在细菌细胞外起作用。Tat系统是不寻常的，因为与大多数其他蛋白质转运体不同，它只运输已经折叠的蛋白质。注定由Tat系统分泌的蛋白质有一个特殊的氨基酸特征序列，一开始被称为“双精氨酸信号”。该信号将蛋白质定向到嵌入在膜中的Tat机制，并促进其分泌。塔塔机器本身由三部分组成：TatA、TatB和TatC。TatA， TatB和TatC成分最初彼此形成1:1:1的复合物这个复合物通过与双精氨酸信号相互作用，负责识别Tat系统分泌的每种不同蛋白质。在信号被TatABC结合后，它触发了TatA成分的额外拷贝，组装成一个环状结构，然后允许蛋白质的运输。在蛋白质运输完毕后，TatA环就会分解，准备进行下一轮的分泌。一些Tat底物蛋白是不寻常的，因为它们在c端含有一个膜相互作用域，将它们固定在膜上。在大肠杆菌中，这些膜固定的Tat底物对于细菌适应不同的生长条件和在哺乳动物感染期间存活是必不可少的。我们感兴趣的是了解Tat途径如何能够将这些蛋白质整合到膜中。我们已经在Tat系统中分离出不能整合这些蛋白质的突变体，而是将它们完全分泌到细胞膜上。我们想了解为什么这些突变体在整合Tat底物蛋白方面存在缺陷。这项工作将帮助我们了解Tat机制如何识别膜蛋白，也可能帮助我们了解Tat机制在运输完成后分解的机制。

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. 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 TatA, TatB and TatC components initially form a 1:1:1 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 TatABC this triggers additional copies of 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.Some of the Tat substrate proteins are unusual because they contain a membrane interaction domain at their C-terminus that anchors them into the membrane. In E. coli these membrane-anchored Tat substrates are essential for the bacterium to adapt to different growth conditions, and to survive during infection of mammals. We are interested in understanding how the Tat pathway is able to integrate these proteins into the membrane. We have isolated mutants in the Tat system that cannot integrate these proteins, instead secreting them completely across the membrane. We want to understand why these mutants are defective in integrating the Tat substrate proteins. This work will help us to understand how the Tat machinery recognises membrane proteins, and may also help us to understand the mechanism by which the Tat machinery disassembles once transport is complete.