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

所有的细胞都被作为屏障的膜包围着。嵌入膜中的蛋白质是营养物质和信息（信号）通过这一屏障的运输所必需的。转运系统需要将蛋白质运送到膜内或穿过膜到达细胞位置，在那里它们可以完成它们的任务。易位系统通过嵌入在氨基酸序列中的信号识别要易位的特定蛋白质。Sec易位系统被很好地研究和保存，从大肠杆菌到人类，突出了它的重要性。因此，关于细菌细胞SecYEG转位机制的发现可以告诉我们类似的系统是如何在我们自己的身体中起作用的。SecYEG是一种膜蛋白复合物，由3个亚基组成。它包含一个通过膜进行蛋白质转运的中心通道。通道也可以在一侧打开，允许蛋白质进入膜的横向通道。在细菌中发现了其他辅助蛋白，以帮助膜蛋白转运和折叠，并将其组装成更大的复合物。尽管这些蛋白质对细胞存活至关重要，但人们对这些蛋白质的工作原理知之甚少。这主要是由于它们与SecYEG易位子形成一个大的组装体，并且很难产生用于实验研究的高阶复合体。通过使用新技术，我们已经成功地产生了大的全息转座子复合物，并建议研究全息转座子中的辅助蛋白如何帮助折叠从核糖体中产生的膜蛋白，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研究这些由膜蛋白合成核糖体和活跃的全息转座子机器组成的分子机器。由于硬件和软件的最新发展，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核糖体和易位折叠膜蛋白的分子相互作用。折叠不正确的蛋白质会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，就会降解折叠错误的蛋白质。细菌膜的两种成分被认为具有关键作用：YidC和FtsH-HflKC复合物。两者也以同源蛋白的形式存在于人类细胞器线粒体中。它们如何工作和识别未折叠的蛋白质目前尚不清楚。我们将在这里阐明这些机器如何协同工作，以确保膜蛋白的适当折叠，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，我们将使用电子显微镜在高分辨率下观察我们在这项工作中识别的相关复合物。因此，我们的研究将为鲜为人知的膜蛋白发生、折叠和伴随的质量控制过程提供重要的新见解。

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.