Periodic Reporting for period 1 - StructureSND (Structural basis for membrane protein biogenesis at the Endoplasmic reticulum by the SND pathway)
Reporting period: 2023-06-01 to 2025-05-31
One-third of eukaryotic genes encode for integral membrane proteins (IMPs) with the majority being assembled at the endoplasmic reticulum (ER) after being translated by the ribosome. The SRP-independent (SND) targeting pathway was recently identified as a new route for ER targeting and insertion of IMPs. It was found to recognise a subset of IMPs with a central transmembrane domain, yet it also serves as an alternative route to deliver a broad range of substrates to the ER when their canonical pathways are impaired. The yeast SND pathway comprises three components, SND1, SND2 and SND3. The cytosolic SND1 is a putative ribosome-associated factor and receptor for nascent IMP substrates. SND2 and SND3 are proposed to form a heterodimeric complex at the ER membrane that, together with the SEC61 translocon, mediates membrane insertion of substrates. However, direct evidence for the roles of the SND components and their physical interactions with each other and complexes such as the ribosome and the SEC61 translocon is lacking.
This project aims to elucidate the molecular interplays between SND components and other targeting and insertion factors using a combination of structural biology, biophysics, and biochemistry. First, a genomic tagging system using a thermophilic fungus, Chaetomium thermophilum, will be established to purify native SND-associated complexes. An integrative analysis using mass spectrometry and different biophysical tools will be subsequently conducted to identify and characterise the interactions between SND components and their associated proteins involved in ER membrane targeting and insertion. The ultimate goal of this study is to determine the structures of endogenous SND complexes by cryo-electron microscopy. This project will provide comprehensive insights into how the SND complexes capture a broad range of substrates and execute their ER membrane targeting and insertion, and will thus have far-reaching impacts on our current understanding of membrane protein biology.
This project aims to elucidate the molecular interplays between SND components and other targeting and insertion factors using a combination of structural biology, biophysics, and biochemistry. First, a genomic tagging system using a thermophilic fungus, Chaetomium thermophilum, will be established to purify native SND-associated complexes. An integrative analysis using mass spectrometry and different biophysical tools will be subsequently conducted to identify and characterise the interactions between SND components and their associated proteins involved in ER membrane targeting and insertion. The ultimate goal of this study is to determine the structures of endogenous SND complexes by cryo-electron microscopy. This project will provide comprehensive insights into how the SND complexes capture a broad range of substrates and execute their ER membrane targeting and insertion, and will thus have far-reaching impacts on our current understanding of membrane protein biology.
To achieve the objectives of this project, we established several genetically modified C. thermophilum strains, which were utilized for the isolation of native SND-associated complexes. We successfully purified a complex that includes SND3, CCDC47, TRAPα and the SEC61 complex. Notably, this complex was found to be tightly associated with the ribosome. We then employed single-particle cryo-electron microscopy (cryo-EM) analysis to determine the structure of this complex. Ultimately, we obtained a cryo-EM structure at 2.2 Å resolution, revealing that this so-called SND3 translocon complex resides beneath the ribosomal tunnel exit. This positioning suggests that SND3 plays a role in co-translational IMP insertion. Through a combination of structural analysis and molecular dynamics (MD) simulations, our findings indicate that SND3 has several hallmark features of a membrane insertase, albeit with a distinct protein topology from the widespread Oxa1 superfamily. Furthermore, we demonstrate that the configuration of the SND3 translocon is analogous to that of the metazoan multipass translocon, which is known to regulate the biogenesis of multipass IMPs. This suggests that the SND3 translocon functions as a reduced-complexity multipass translocon, regulating the co-translational insertion of multipass IMPs in fungi and other lower eukaryotes.
This work has been deposited on a preprint server and shared on social media. It is currently undergoing peer review in an open-access journal.
This work has been deposited on a preprint server and shared on social media. It is currently undergoing peer review in an open-access journal.
Due to their critical involvement in diverse physiological roles (e.g. transport, signaling and catalysis) IMPs represent nearly 60% of clinical drug targets. It is therefore important to understand IMP biogenesis, which mostly starts with targeting and insertion at the ER. Three ER biogenesis pathways have been well characterized by structural biology and other methods, however many IMPs are still known to be targeted by independent routes. The discovery of the SND pathway provided a crucial missing puzzle piece, delivering a distinct subset of IMPs to the ER and acting as a backup when other canonical targeting pathways are impaired. However, the SND pathway remains mechanistically unclear, and each SND component has yet to be fully characterized in terms of their structure and function. In our project, we used a combination of cryo-EM analysis, MD simulations and mass spectrometry-based proteomics to reveal that one of the SND components, SND3, is involved in a co-translational machinery. Our results demonstrate that SND3 serves as a membrane insertase within a newly identified SEC61 translocon complex associated with the ribosome, which we have named the SND3 translocon. Our findings suggest that the SND3 translocon plays a crucial role in regulating the biogenesis of multipass membrane proteins in fungi and other eukaryotic lineages (including parasitic Trypanosoma and Leishmania spp.) that do not possess a metazoan-like multipass translocon. Given that SND3 has a novel fold for a membrane insertase, it could further serve as an attractive drug target in these organisms.