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

• 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

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.

所有的细胞都被膜所包围，这些膜起着屏障的作用。嵌入膜中的蛋白质是营养物质和信息（信号）穿过这一屏障所必需的。需要转运系统将蛋白质转运到膜中或穿过膜到达它们可以完成其任务的细胞位置。易位系统通过嵌入在构建它们的氨基酸序列中的信号识别待易位的特定蛋白质。Sec易位系统被很好地研究，并且从细菌大肠杆菌到人类都是保守的，突出了其重要性。因此，关于细菌细胞中SecYEG易位机制的发现可以告诉我们类似的系统如何在我们自己的身体中工作。SecYEG是包含3个亚基的膜蛋白复合物。它含有一个中央通道，用于蛋白质转运通过膜。通道也可以在侧面打开，以允许蛋白质进入膜的侧向通道。在细菌中发现了额外的辅助蛋白，以帮助膜蛋白移位和折叠，并将它们组装成更大的复合物。尽管这些蛋白质对细胞生存至关重要，但对这些蛋白质如何工作的了解要少得多。这主要是由于它们与SecYEG易位子形成大的组装体，并且难以产生用于实验研究的这种高阶复合物。通过使用新技术，我们已经成功地产生了大的全息-转位子复合物，并在这里建议研究全息-转位子中的辅助蛋白如何帮助折叠从核糖体中出现的膜蛋白，核糖体是细胞的蛋白质工厂。为此，我们将通过最先进的电子显微镜和图像处理直接观察这些分子机器，包括膜蛋白合成核糖体和活性全转位子机器。由于硬件和软件的最新发展，该技术现在能够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以及易位的待折叠膜蛋白的分子相互作用。未正确折叠的蛋白质由细胞质量控制系统识别。该系统首先尝试在能量的帮助下折叠蛋白质，如果不成功，则降解错误折叠的蛋白质。细菌膜的两种组分被认为具有关键作用：YidC和FtsH-HflKC复合物。这两种蛋白质也以同源蛋白质的形式存在于人类细胞器中，即细胞器。它们如何工作和识别未折叠的蛋白质今天还不清楚。我们将在这里阐明这些机器如何协同工作，以确保膜蛋白的正确折叠，并去除可能对细胞有害的未折叠蛋白质。我们将使用纯化的组分来生物化学地剖析折叠/降解机器的相互作用和机制，并且我们将使用电子显微镜以高分辨率可视化我们在这项工作中识别的相关复合物。因此，我们的研究将提供必要的新的见解知之甚少的膜蛋白的发生，折叠过程，并伴随着质量控制。

项目成果

Multiprotein Complex Production in E. coli: The SecYEG-SecDFYajC-YidC Holotranslocon.

大肠杆菌中的多蛋白复合物生产：SecYEG-SecDFYajC-YidC Holotranslocon。

• DOI: 10.1007/978-1-4939-6887-9_18
• 发表时间: 2017
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Berger I

Structure and Dynamics of the Central Lipid Pool and Proteins of the Bacterial Holo-Translocon.

细菌全息子中心脂质池和蛋白质的结构和动力学。

• DOI: 10.1016/j.bpj.2019.04.002
• 发表时间: 2019
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Martin R

The SARS-CoV-2 spike protein: balancing stability and infectivity.

• DOI: 10.1038/s41422-020-00430-4
• 发表时间: 2020-12
• 期刊: Cell research
• 影响因子: 44.1
• 作者: Berger I;Schaffitzel C
• 通讯作者: Schaffitzel C

Efficient production of a mature and functional gamma secretase protease.

• DOI: 10.1038/s41598-018-30788-w
• 发表时间: 2018-08-27
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Khan I;Krishnaswamy S;Sabale M;Groth D;Wijaya L;Morici M;Berger I;Schaffitzel C;Fraser PE;Martins RN;Verdile G
• 通讯作者: Verdile G

Structure and dynamics of the central lipid pool and protein components of the bacterial holo-translocon

细菌全息子的中心脂质池和蛋白质成分的结构和动力学

• DOI: 10.1101/490250
• 发表时间: 2018
• 作者: Martin R