Functional ER architecture

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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.