Protein ER-ways · Protein ER-ways: assembling ER-translocons on-the fly

The human proteome is primarily synthesized at the endoplasmic reticulum (ER) membrane through the ribosome-translocon complex. This assembly is crucial for protein synthesis, translocation, membrane insertion and post-translational modification. Depending on the type of protein being synthesized, the translocon adopts either a secretory (soluble proteins) or multipass (membrane proteins) form. Newly synthesized proteins often carry a signal peptide that is cleaved by the signal peptidase complex (SPC) co-translationally, indicating possible structural interactions between the ribosome-translocon complex and the SPC during this process. In the proposed project, I aim to elucidate these interactions and explore how different translocon variant recruitment affects polysome topology, as well as reveal the structural interactions between the ribosome-translocon complex and the SPC during signal-peptide cleavage in high-resolution. I will carry out the project in three phases. First, I will develop a multimodal mRNA probe to detect ribosome-translocon-SPC complexes, enabling their visualization during protein synthesis. In the second phase I will investigate how different translocon variants affect polysome topology using advanced imaging techniques such as cryo-correlative light electron microscopy (cryo-CLEM) and cryo-electron tomography (cryo-ET) ex vivo. These methods will help identify how polysome topology is affected by recruitment of specific translocon variants interact at the ER membrane. Finally, I will use single-particle cryo-electron microscopy (SPA cryo-EM) to achieve a high-resolution reconstruction of the ribosome-translocon-SPC complex, providing detailed insights into the molecular interactions during nascent polypeptide synthesis and signal peptide cleavage. This research outcomes of the proposed project will significantly advance our understanding of ER protein biogenesis and its regulatory mechanisms.