Membrane protein insertion and quality control by the bacterial holo-translocon and FtsH chaperone/protease complex

通过细菌全息子和 FtsH 伴侣/蛋白酶复合物进行膜蛋白插入和质量控制

基本信息

批准号：
BB/P000940/1
负责人：
Christiane Berger-Schaffitzel
金额：
$ 51.37万
依托单位：
University of Bristol
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FP000940%2F1
关键词：
Membrane protein insertion quality control

项目摘要

All cells are surrounded by membranes that act as a barrier. Proteins embedded in the membrane are required for the transport of nutrients and information (signals) across this barrier. Translocation systems are required to transport proteins into the membrane or across the membrane to the cellular location where they can fulfil their tasks. Translocation systems recognise the specific proteins to be translocated via signals embedded in the sequence of amino acids from which they are constructed. The Sec translocation system is well studied and conserved from the bacterium Escherichia coli to humans highlighting its importance. Findings about the mechanism of the SecYEG translocon from bacterial cells can thus inform us how similar systems work in our own bodies. SecYEG is a membrane protein complex that comprises 3 subunits. It contains a central channel for protein translocation through the membrane. The channel can also open up on the side to allow the lateral passage of proteins into the membrane. Additional accessory proteins were identified in bacteria to help translocating and folding the membrane proteins and to assemble them into larger complexes. Despite the fact that these proteins are essential for cell survival, much less is known about how these proteins work. This is mostly due to the fact that they form a large assembly with the SecYEG translocon and it was difficult to produce this higher-order complex for experimental studies. By using new technology we have succeeded in generating the large holo-translocon complex and suggest here to study how the accessory proteins in the holo-translocon help to fold up membrane proteins that emerge from the ribosome, which is the protein factory of the cell. To this end, we will directly look at these molecular machines consisting of membrane-protein synthesizing ribosomes and active holo-translocon machinery by state-of-the-art electron microscopy and image processing. Due to recent developments in hard and software, this technology now enables to obtain unprecedented high-resolution structures and thus insights into the molecular interplay of translocation proteins with the synthesizing ribosome and with the translocated, to be folded membrane protein.Proteins that are not correctly folded are recognized by the cellular quality control system. This system first attempts to fold up the protein with the help of energy, and if unsuccessful, degrades the mis-folded protein. Two components of the bacterial membrane are thought to have a key role: YidC and the FtsH-HflKC complex. Both are also present as homologous proteins in a human cellular organelle, the mitochondrion. How they work and recognize the unfolded protein is unknown today. We will elucidate here how these machines work together to ensure the proper folding of membrane proteins and to remove unfolded proteins that could be detrimental to the cell. We will use purified components to biochemically dissect the interplay and the mechanism of the folding/degradation machine, and we will use electron microscopy to visualize at high resolution the relevant complexes which we identify in this work. Our studies will thus provide essential new insights into the poorly understood process of membrane protein genesis, folding, and concomitant quality control.

所有细胞都被充当屏障的膜包围。嵌入在膜上的蛋白质是跨该屏障的养分和信息（信号）运输所必需的。需要易位系统将蛋白质运输到膜或整个膜上到可以完成任务的细胞位置。易位系统识别特定的蛋白质通过嵌入中氨基酸序列中的信号易位的特定蛋白质。从细菌大肠杆菌到人类，对SEC易位系统进行了充分的研究和保守，强调了其重要性。因此，关于细菌细胞的Secyeg易位机理的发现可以告知我们类似系统如何在我们自己的身体中起作用。 Secyeg是一种膜蛋白复合物，包括3个亚基。它包含通过膜易位的中心通道。该通道还可以在侧面打开，以使蛋白质横向传递到膜中。在细菌中鉴定出其他辅助蛋白，以帮助易位和折叠膜蛋白，并将它们组装成较大的络合物。尽管这些蛋白质对于细胞存活至关重要，但对于这些蛋白质的起作用而言，知之甚少。这主要是由于它们与secyeg cllessocon形成大型组装，并且很难为实验研究产生这种高阶复合物。通过使用新技术，我们成功地生成了大型全晶型晶体复合物，并在这里建议研究Holo-Translocon中的辅助蛋白如何有助于折叠从核糖体中出现的膜蛋白，这是细胞的蛋白质工厂。为此，我们将直接查看这些分子机器，这些计算机包括膜 - 蛋白质合成的核糖体和通过最新的电子显微镜和图像处理的主动全透透明机械。由于硬性和软件的最新发展，这项技术现在使得可以获得前所未有的高分辨率结构，从而深入了解易位蛋白与合成核糖体的分子相互作用，并与转运的膜蛋白折叠。蛋白质不正确折叠的蛋白质由蜂窝质量控制系统识别。该系统首先尝试借助能量折叠蛋白质，如果失败，则会降解折叠式蛋白质的蛋白质。细菌膜的两个组成部分被认为具有关键作用：YIDC和FTSH-HFLKC复合物。两者在人类细胞细胞器（线粒体）中也以同源蛋白的形式存在。他们的工作方式和识别未折叠的蛋白质今天尚不清楚。我们将在这里阐明这些机器如何共同工作，以确保膜蛋白的正确折叠并去除可能对细胞有害的展开蛋白质。我们将使用纯化的组件来剖析折叠/降解机的相互作用和机理，我们将使用电子显微镜在高分辨率下可视化我们在这项工作中识别的相关复合物。因此，我们的研究将提供对膜蛋白发生，折叠和随之而来的质量控制过程不佳的知识过程的重要新见解。