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.

所有细胞都被充当屏障的膜包围。穿过该屏障运输营养物质和信息（信号）需要嵌入膜中的蛋白质。易位系统需要将蛋白质转运到膜内或跨膜至它们可以完成其任务的细胞位置。易位系统通过嵌入氨基酸序列中的信号来识别要易位的特定蛋白质。 Sec 易位系统从大肠杆菌到人类都得到了充分研究和保守，凸显了其重要性。因此，关于细菌细胞 SecYEG 易位子机制的发现可以告诉我们类似的系统如何在我们自己的体内发挥作用。 SecYEG 是一种膜蛋白复合物，包含 3 个亚基。它包含一个蛋白质通过膜易位的中央通道。该通道还可以在侧面打开，以允许蛋白质横向进入膜。在细菌中发现了额外的辅助蛋白，以帮助易位和折叠膜蛋白并将它们组装成更大的复合物。尽管这些蛋白质对于细胞生存至关重要，但人们对这些蛋白质如何发挥作用却知之甚少。这主要是因为它们与 SecYEG 易位子形成了一个大的组装体，并且很难产生这种高阶复合物用于实验研究。通过使用新技术，我们成功地生成了大型全息易位子复合物，并在此建议研究全息易位子中的辅助蛋白如何帮助折叠从核糖体（细胞的蛋白质工厂）中产生的膜蛋白。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由膜蛋白合成核糖体和活性全息子机器组成的分子机器。由于最近硬件和软件的发展，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、待折叠膜蛋白的分子相互作用。细胞质量控制系统可以识别未正确折叠的蛋白质。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 复合物。两者也作为同源蛋白质存在于人类细胞器线粒体中。如今，它们如何工作并识别未折叠的蛋白质尚不清楚。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。我们将使用纯化的成分来生化剖析折叠/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，我们的研究将为人们对膜蛋白发生、折叠和伴随的质量控制知之甚少的过程提供重要的新见解。

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