Structural analysis of the dynamic ribosome-translocon complex

Reviewer #1 (Public Review):

The paper 'Structural Analysis of the Dynamic Ribosome-Translocon Complex,' authored by Lewis et al., meticulously explores various conformations and states of the ribosome-translocon complex. Employing advanced techniques such as cryoEM structural determination and AlphaFold modeling, the study delves into the dynamic nature of the ribosome-translocon complex. The findings from these analyses unveil crucial insights, significantly advancing our understanding of the co-translational translocation process in cellular mechanisms.

To begin with, the authors employed a construct comprising the first two transmembrane domains of rhodopsin as a model for studying protein translocation. They conducted in vitro translation, followed by the purification of the ribosome-translocon complex, and determined its cryoEM structures. An in-depth analysis of their ribosome-translocon complex structure revealed that the nascent chain can pass through the lateral gate of translocon Sec61, akin to the behavior of a Signaling Peptide. Additionally, Sec61 was found to interact with 28S rRNA helix 24 and the ribosomal protein uL24. In summary, their structural model aligns with the through-pore model of insertion, contradicting the sliding model.

Secondly, the authors successfully identified RAMP4 in their ribosome-translocon complex structure. Notably, the transmembrane domain of RAMP4 mimics the binding of a Signaling Peptide at the lateral gate of Sec61, albeit without unplugging. Intriguingly, RAMP4 is exclusively present in the non-multipass translocon ribosome-translocon complex, not in those containing multipass translocon. This observation suggests that co-translational translocation specifically occurs in the Sec61 channel that includes bound RAMP4. Additionally, the authors discovered an interaction between the C-tail of ribosomal proteins uL22 and the translocon Sec61, providing valuable insights into the nascent chain's behavior.

Moving on to the third point, the focused classification unveiled TRAP complex interactions with various components. The authors propose that the extra density observed in their novel ribosome-translocon complex can be attributed to calnexin, a major binder of TRAP according to previous studies. Furthermore, the new structure reveals a TRAP-OSTA interaction. This newly identified TRAP-OSTA interaction offers a potential explanation for why patients with TRAP delta defects exhibit congenital disorders of glycosylation.

In conclusion, this paper presents a robust contribution to the field with its thorough structural and modeling analyses. The significance of the findings is evident, providing valuable insights into the intricate mechanisms of protein co-translational translocation. The well-crafted writing, meticulous analyses, and clear figures collectively contribute to the overall strength of the paper.

Major points:

(1) The identification of RAMP4 is a pivotal discovery in this paper. The sophisticated AlphaFold prediction, de novo model building of RAMP4's RBD domain, and sequence analyses provide strong evidence supporting the inclusion of RAMP4 in the ribosome-translocon complex structure.

However, it is crucial to ensure the presence of RAMP4 in the purified sample. Particularly, a validation step such as western blotting for RAMP4 in the purified samples would strengthen the assertion that the ribosome-translocon complex indeed contains RAMP4. This is especially important given the purification steps involving stringent membrane solubilization and affinity column pull-down.

(2) Despite the comprehensive analyses conducted by the authors, it is challenging to accept the assertion that the extra density observed in TRAP class 1 corresponds to calnexin. The additional density in TRAP class 1 appears to be less well-resolved, and the evidence for assigning it as calnexin is insufficient. The extra density there can be any proteins that bind to TRAP. It is recommended that the authors examine the density on the ER lumen side. An investigation into whether calnexin's N-globular domain and P-domain are present in the ER lumen in TRAP class 1 would provide a clearer understanding.

(3) In the section titled 'TRAP competes and cooperates with different translocon subunits,' the authors present a compelling explanation for why TRAP delta defects can lead to congenital disorders of glycosylation. To enhance this explanation, it would be valuable if the authors could provide additional analyses based on mutations mentioned in the references. Specifically, examining whether these mutations align with the TRAP delta-OSTA structure models would strengthen the link between TRAP delta defects and the observed congenital disorders of glycosylation.

Reviewer #2 (Public Review):

Summary:

In the manuscript 'Structural analysis of the dynamic ribosome-translocon complex' Lewis and Hegde present a structural study of the ribosome-bound multipass translocon (MPT) based on re-analysis of cryo-EM single particle data of ribosome-MPTs processing the multipass transmembrane substrate RhoTM2 from a previous publication (Smalinskaité et al, Nature 2022) and AlphaFold2 multimer modeling. Detailed analysis of the laterally open Sec61 is obtained from PAT-less particles.

The following major claims are made:

- TMs can bind similarly to the Sec61 lateral gate as signal peptides.

- Ribosomal H59 is in immediate proximity to basic residues of TMs and signal peptides, suggesting it may contribute to the positive-inside rule.

- RAMP4/SERP1 binds to the Sec61 lateral gate and the ribosome near 28S rRNA's helices 47, 57, and 59 as well as eL19, eL22, and eL31.

- uL22 C-terminal tail binds H24/47 blocking a potential escape route for nascent peptides to the cytosol.

- TRAP and BOS compete for binding to Sec61 hinge.

- Calnexin TM binds to TRAPg.

- NOMO wedges between TRAP and MPT.

Strengths:

The manuscript contains numerous novel new structural analyses and their potential functional implications. While all findings are exciting, the highlight is the discovery of RAMP4/SERP1 near the Sec61 lateral gate. Overall, the strength is the thorough and extensive structural analysis of the different high-resolution RTC classes as well as the expert bioinformatic evolutionary analysis.

Weaknesses:

A minor downside of the manuscript is the sheer volume of analyses and mechanistic hypotheses, which makes it sometimes difficult to follow. The authors might consider offloading some analyses based on weaker evidence to the supplement to maximize impact.

Author response:

Factual error in the eLife assessment to be corrected:

In the eLife assessment, "ribosomal protein H59" should be changed to "helix 59 of the 28S ribosomal RNA" to make this factually correct.

Provisional author response

We thank the reviewers for their thorough and thoughtful readings of the manuscript. Our responses to the four suggestions made in their public reviews are below.

Reviewer #1 (Public Review):

Major points:

(1) The identification of RAMP4 is a pivotal discovery in this paper. The sophisticated AlphaFold prediction, de novo model building of RAMP4's RBD domain, and sequence analyses provide strong evidence supporting the inclusion of RAMP4 in the ribosome-translocon complex structure.

However, it is crucial to ensure the presence of RAMP4 in the purified sample. Particularly, a validation step such as western blotting for RAMP4 in the purified samples would strengthen the assertion that the ribosome-translocon complex indeed contains RAMP4. This is especially important given the purification steps involving stringent membrane solubilization and affinity column pull-down.

As suggested, we will revise the manuscript to include Western blots showing that RAMP4 is retained at secretory translocons (and not multipass translocons) after solubilisation, affinity purification, and recovery of ribosome-translocon complexes.

(2) Despite the comprehensive analyses conducted by the authors, it is challenging to accept the assertion that the extra density observed in TRAP class 1 corresponds to calnexin. The additional density in TRAP class 1 appears to be less well-resolved, and the evidence for assigning it as calnexin is insufficient. The extra density there can be any proteins that bind to TRAP. It is recommended that the authors examine the density on the ER lumen side. An investigation into whether calnexin's N-globular domain and P-domain are present in the ER lumen in TRAP class 1 would provide a clearer understanding.

We agree that the Calnexin assignment is less confident than the other assignments in this manuscript, and that further support would be ideal. We have exhaustively searched our maps for any unexplained density connected with the putative Calnexin TMD, and have found none. This is consistent with Calnexin's lumenal domain being flexibly linked to its TMD, and thus would not be resolved in a ribosome-aligned reconstruction.

Our assignment of this TMD to Calnexin was based on existing biochemical data (referenced in the paper) favouring this as the best working hypothesis by far: Calnexin is TRAP’s only abundant co-purifying factor, and their interaction is sensitive to point mutations in the Calnexin TMD. Recognising that this is not conclusive, we will ensure that the text and figures consistently describe this assignment as provisional or putative.

(3) In the section titled 'TRAP competes and cooperates with different translocon subunits,' the authors present a compelling explanation for why TRAP delta defects can lead to congenital disorders of glycosylation. To enhance this explanation, it would be valuable if the authors could provide additional analyses based on mutations mentioned in the references. Specifically, examining whether these mutations align with the TRAP delta-OSTA structure models would strengthen the link between TRAP delta defects and the observed congenital disorders of glycosylation.

We agree that mapping disease-causing point mutants to the TRAP delta structure could be potentially informative. Unfortunately, the referenced TRAP delta disease mutants act by simply impairing TRAP delta expression, and thus admit no such fine-grained analyses. However, sequence conservation is our next best guide to mutant function. We note in the text that the contact site charges on TRAP delta and RPN2 are conserved, and that the closest-juxtaposed interaction pair (K117 on TRAPδ and D386 on RPN2) is also the most conserved.

Reviewer #2 (Public Review):

Strengths:

The manuscript contains numerous novel new structural analyses and their potential functional implications. While all findings are exciting, the highlight is the discovery of RAMP4/SERP1 near the Sec61 lateral gate. Overall, the strength is the thorough and extensive structural analysis of the different high-resolution RTC classes as well as the expert bioinformatic evolutionary analysis.

Weaknesses:

A minor downside of the manuscript is the sheer volume of analyses and mechanistic hypotheses, which makes it sometimes difficult to follow. The authors might consider offloading some analyses based on weaker evidence to the supplement to maximize impact.

We agree that the manuscript is long, and we will seek ways to streamline it in revision while avoiding the undesirable side effect of making important findings undiscoverable via literature searches (an unfortunate consequence of many supplemental data). Indeed, we chose eLife for its flexibility regarding article length and suitability for extended and detailed analyses.