Visualizing Protein Biogenesis at the ER Membrane

The ribosome-associated endoplasmic reticulum (ER) translocon machinery is the entry point of the secretory pathway and governs translocation, signal peptide (SP) insertion and cleavage, multispanning membrane protein insertion, N-glycosylation, glycoprotein processing, and protein folding and assembly. To specialize for translocation of specific protein subsets, various accessory factors need to associate with the translocon. Recently, cryo-electron microscopy (cryo-EM) provided valuable insights into the structure and function of many translocon-bound factors, however, single-particle cryo-EM approaches are often associated with detergent-solubilization and purification of the target factors, which may introduce artefacts or disrupt interactions to weak binding partners. Cryo-electron tomography (cryo-ET) studies, which do not rely on extensive purification procedures and enable visualization of protein complexes in their native membrane environment, remain scarce. This thesis aims to explore the close-to-native molecular landscape of the ribosome-associated ER translocon. Using cryo-ET and extensive subtomogram analysis, I revealed eight actively translating ribosome intermediates, seven of which I assigned to the elongation cycle, as well as two hibernating ribosome states. I identified a previously unknown intermediate associated with elongation factor 1a (eEF1a) in the extended conformation, indicating that eEF1a remains bound to the ribosome after GTP-hydrolysis and tRNA accommodation and possibly contributes to proofreading in mRNA decoding. Using subtomogram analysis, I explored the landscape of the ER translocon and identified three major variants, the Sec61-TRAP, the Sec61-TRAP-OSTA, and the Sec61-multipass translocon. Tomogram segmentation and spatial neighborhood analysis demonstrate that OSTA as well as multipass variants cluster in distinct polysome chains, reflecting their substrate specificity for SP-containing and multispanning membrane proteins, respectively. Based on the reconstruction of Sec61-TRAP-OSTA and ColabFold prediction models, we built a near-complete molecular model of this ER translocon, revealing the molecular details of TRAP and its interactions with Sec61 and an unidentified OSTA-specific transmembrane protein named T1. Reconstructions of the OSTA feature a SP-like density, which likely represents an unidentified translocon component, possibly RAMP4, and potentially plays a pivotal role in regulation of translocation and topogenesis. In the ER lumen, I found additional densities, named L1 and L2, associated with OSTA complex. Preliminary data suggest that PDIA5 is a prime candidate for L2 and likely enhances glycoprotein folding at the translocon. Moreover, I characterized the composition of the multipass translocon in context of translational activity. TRAP, a novel accessory factor of the multipass translocon, and PAT, an intramembrane chaperone for multispanning transmembrane domains, are variably recruited to the multipass translocon. Cryo-ET data of ER-derived vesicles under steady state and ER stress condition suggest that PAT preferentially associates with the actively translocating multipass translocon, while TRAP is preferentially recruited to the inactive variant. Moreover, refinement of the Sec61-TRAP-multipass translocon visualizes interactions between TRAP and an unidentified protein, most likely BOS subunit NOMO, which may be important for intermolecular communication during multipass membrane protein biogenesis. Using cryo-ET, I dissect ribosomal intermediate states, visualize ER translocon variants and novel interaction partners, and characterize their organization at the ER membrane. These results advance our understanding of various protein biogenesis processes and provide a structural basis for future investigations.