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.

所有细胞都被起到屏障作用的膜所包围。膜中嵌入的蛋白质是营养物质和信息(信号)通过这一屏障的传输所必需的。转运系统需要将蛋白质运输到细胞膜或跨膜到细胞位置，在那里它们可以完成它们的任务。转位系统通过嵌入氨基酸序列中的信号识别要转位的特定蛋白质，这些蛋白质是由氨基酸组成的。SEC易位系统从大肠杆菌到人类都得到了很好的研究和保守，强调了它的重要性。因此，关于细菌细胞中SecYEG转位机制的发现可以告诉我们，类似的系统在我们的身体中是如何工作的。SecYEG是由3个亚基组成的膜蛋白复合体。它包含一个中央通道，用于蛋白质通过膜的转运。该通道还可以在侧面打开，以允许蛋白质侧向进入膜。在细菌中发现了更多的辅助蛋白，以帮助膜蛋白的移位和折叠，并将它们组装成更大的复合体。尽管这些蛋白质对细胞生存是必不可少的，但人们对这些蛋白质是如何工作的知之甚少。这主要是因为它们与SecYEG转运子形成了一个大的组合体，很难产生这种用于实验研究的高阶复合体。通过使用新技术，我们成功地产生了大的全转位复合体，并建议在这里研究全转位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技术直接观察这些由膜蛋白合成核糖体和活性全位转位组成的分子机器。由于最近在硬件和软件方面的发展，这项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的核糖体以及与易位的折叠的膜蛋白之间的分子相互作用。没有正确折叠的蛋白质被细胞质量控制系统识别。这个系统首先尝试在能量的帮助下折叠蛋白质，如果不成功，就会降解错误折叠的蛋白质。细菌膜的两个成分被认为具有关键作用：YidC和FtsH-HflKC复合体。这两种蛋白质也都以同源蛋白质的形式存在于人类细胞器--线粒体中。它们是如何工作并识别未折叠的蛋白质的，目前尚不清楚。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。我们将使用纯化的组分来生化剖析折叠/降解机器的相互作用和机制，并使用电子显微镜以高分辨率显示我们在这项工作中确定的相关络合物。因此，我们的研究将对膜蛋白发生、折叠和伴随的质量控制这一鲜为人知的过程提供必要的新见解。

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