Functional ER architecture

在所有真核细胞中，新生蛋白和分泌蛋白都必须跨内质网（ER）膜转运或整合到内质网（ER）膜上，这些蛋白是注定要存在于胞内和胞外途径细胞器的膜和管腔中的。在哺乳动物中，大多数蛋白质在胞质核糖体合成时是易位的（共翻译），而许多对细胞间通讯和调节至关重要的小分泌前蛋白质是翻译后输入的。在这个联合项目中，我们建议从组成、结构和成分功能方面描述哺乳动物多肽在翻译中和翻译后易位到内质网及其伴随的共价修饰的蛋白质机制。为了研究大约40个不同亚单位的结构和相互作用，我们使用了一种公正的、高度跨学科的方法。系统sirna介导的人体细胞易位机制组件敲除和基于质谱（MS）的定量蛋白质组学将用于鉴定和分类它们的特定底物。利用半透性和完整的人类细胞对已建立的和新鉴定的底物进行易位试验，将有助于在siRNA沉默和与突变cDNA互补的帮助下，对易位机制组件进行深入的功能表征。低温电子断层扫描（CET）结合亚层析成像平均将首先提供天然共平移易位机制（共易位）核心的三维地图。一些亚基将在这种密度的背景下通过比较重建来绘制，其中它们要么融合到适当的功能惰性标签上，要么使用sirna耗尽，而另一些则基于它们的高分辨率x射线晶体学结构。考虑到这些数据以及化学交联和质谱对蛋白质接近度的限制，我们将使用计算模型确定核心共转座子最可能的分子结构。对共易位粒子进行广泛的亚层析成像分类，将揭示围绕核心复合体的共转译易位机制的整个组成景观。最后，我们将使用CET来研究模型底物的翻译后导入，以表征后易位子的结构及其与共易位子的差异。我们发表的和初步的数据已经证明了这里建议的综合方法的可行性。对共译蛋白和翻译后蛋白在内质网膜上的易位和整合以及伴随的共价修饰的结构和机制的了解可能提供对几种人类疾病的病理的详细了解，包括多囊性肝病、糖尿病和各种肿瘤，并导致新的治疗策略。

In all eukaryotic cells nascent proteins that are destined to reside in membranes and lumens of organelles of the endo- and exocytotic pathways, as well as secreted proteins must be translocated across or integrated into the Endoplasmic Reticulum (ER) membrane. In mammals, most proteins are translocated concomitant with their synthesis by cytosolic ribosomes (co-translationally), whereas many small presecretory proteins, which are essential for intercellular communication and regulation, are post-translationally imported. In this joint project, we propose to characterize the mammalian protein machinery for the co- and post-translational translocation of polypeptides into the ER and their accompanying covalent modifications in terms of composition, structure, and component functions. To study the architecture and interplay of the approximately 40 different subunits we use an unbiased, highly interdisciplinary approach. Systematic siRNA-mediated knock down of components of the translocation machinery in human cells and mass-spectrometry (MS) based quantitative proteomics will be used to identify and categorize their specific substrates. Assays for translocation of established and newly identified substrates using both, semi-permeabilized and intact human cells will enable an in-depth functional characterization of the translocation machinery components with the help of siRNA silencing and complementation with mutated cDNA. Cryo¬-electron tomography (CET) in conjunction with subtomogram averaging will provide a three-dimensional map firstly of the core of the native co-translational translocation machinery (co-translocon). Some subunits will be mapped in the context of this density by comparison to reconstructions in which they are either fused to appropriate functionally inert labels or depleted using siRNAs, and others based on their high-resolution X-ray crystallographic structures. Given these data and restraints on protein proximities obtained by chemical cross-linking and MS we will determine the most probable molecular architecture of the core co-translocon using computational modeling. Extensive subtomogram classification of the co-translocon particles will then unravel the entire compositional landscape of the co-translational translocation machinery surrounding the core complex. Finally, we will use CET to study post-translational import of model substrates to characterize the structure of the post-translocon and its differences to the co-translocon. Our published and preliminary data have demonstrated the feasibility of the integrative approach suggested here. The obtained structural and mechanistic insights into co- and post-translational protein translocation across and integration into the ER membrane and the accompanying covalent modifications may provide a detailed understanding of the pathology of several human diseases, including polycystic liver disease, diabetes and various tumors and lead to novel therapeutic strategies.