Biological Sciences Division
Published June 18, 2024 | Version v1
Journal article Open

Structural analysis of the dynamic ribosome-translocon complex

Creators

  • 1. MRC Laboratory of Molecular Biology
  • 2. University of Chicago

Description

The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane proteins. Here, we leveraged recent advances in cryo-electron microscopy (cryo-EM) and structure prediction to derive insights into several novel configurations of the ribosome-translocon complex. We show how a transmembrane domain (TMD) in a looped configuration passes through the Sec61 lateral gate during membrane insertion; how a nascent chain can bind and constrain the conformation of ribosomal protein uL22; and how the translocon-associated protein (TRAP) complex can adjust its position during different stages of protein biogenesis. Most unexpectedly, we find that a large proportion of translocon complexes contains RAMP4 intercalated into Sec61's lateral gate, widening Sec61's central pore and contributing to its hydrophilic interior. These structures lead to mechanistic hypotheses for translocon function and highlight a remarkably plastic machinery whose conformations and composition adjust dynamically to its diverse range of substrates.

Data availability

The EM maps have been deposited in EMDB with the accession numbers EMD-19195, EMD-19196, EMD-19197, EMD-19198, 19199, EMD-19200, EMD-19201, EMD-19202, EMD-19203, and EMD-19204. The molecular models have been deposited in the PDB with accession numbers 8RJB, 8RJC, and 8RJD. The AlphaFold2-predicted structures (Varadi et al., 2024) reported in this study have been deposited in ModelArchive with accession numbers ma-jjnuw, ma-hbsof, ma-hknzh, ma-x3uvj, ma-j8wag, ma-9gsmk, ma-l9qqq, and ma-ise4t.

The following datasets were generated:

Lewis AJO Hegde RS (2024) Electron Microscopy Data Bank ID EMD-19195. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-RAMP4. https://www.ebi.ac.uk/emdb/EMD-19195

Lewis AJO Hegde RS (2024) Electron Microscopy Data Bank ID EMD-19196. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61, all-particle reconstruction. https://www.ebi.ac.uk/emdb/EMD-19196

Lewis AJO Hegde RS (2024) Electron Microscopy Data Bank ID EMD-19197. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, open conformation 1. https://www.ebi.ac.uk/emdb/EMD-19197

Lewis AJO Hegde RS (2024) Electron Microscopy Data Bank ID EMD-19198. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, open conformation 2. https://www.ebi.ac.uk/emdb/EMD-19198

Lewis AJO Hegde RS (2024) Electron Microscopy Data Bank ID EMD-19199. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61, bound TRAP-alpha TMD. https://www.ebi.ac.uk/emdb/EMD-19199

Lewis AJO Hegde RS (2024) Electron Microscopy Data Bank ID EMD-19200. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, conformation 1. https://www.ebi.ac.uk/emdb/EMD-19200

Lewis AJO Hegde RS (2024) Electron Microscopy Data Bank ID EMD-19201. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, conformation 2. https://www.ebi.ac.uk/emdb/EMD-19201

Lewis AJO Hegde RS (2024) Electron Microscopy Data Bank ID EMD-19202. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61, closed conformation 1. https://www.ebi.ac.uk/emdb/EMD-19202

Lewis AJO Hegde RS (2024) Electron Microscopy Data Bank ID EMD-19203. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61, closed conformation 2. https://www.ebi.ac.uk/emdb/EMD-19203

Lewis AJO Hegde RS (2024) Electron Microscopy Data Bank ID EMD-19204. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Rho-bound Sec61. https://www.ebi.ac.uk/emdb/EMD-19204

Lewis AJO Hegde RS (2024) RCSB Protein Data Bank ID 8RJB. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-RAMP4. https://www.rcsb.org/structure/8RJB

Lewis AJO Hegde RS (2024) RCSB Protein Data Bank ID 8RJC. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, open conformation 1. https://www.rcsb.org/structure/8RJC

Lewis AJO Hegde RS (2024) RCSB Protein Data Bank ID 8RJD. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, open conformation 2. https://www.rcsb.org/structure/8RJD

Lewis AJO Hegde RS (2024) ModelArchive ID ma-jjnuw. Chlamydomonas reinhardtii Sec61-RAMP4 complex. https://modelarchive.org/doi/10.5452/ma-jjnuw

Lewis AJO Hedge RS (2024) ModelArchive ID ma-hbsof. Saccharomyces cerevisiae Sec61-RAMP4 complex (aka Ysy6p). https://modelarchive.org/doi/10.5452/ma-hbsof

Lewis AJO Hegde RS (2024) ModelArchive ID ma-hknzh. Canis lupus familiaris Sec61-RAMP4 complex. https://modelarchive.org/doi/10.5452/ma-hknzh

Lewis AJO Hegde RS (2024) ModelArchive ID ma-x3uvj. Heimdallarchaean TRAPα-SecY complex. https://modelarchive.org/doi/10.5452/ma-x3uvj

Lewis AJO Hegde RS (2024) ModelArchive ID ma-j8wag. Trypanosoma brucei TRAP-Sec61 complex. https://modelarchive.org/doi/10.5452/ma-j8wag

Lewis AJO Hegde RS (2024) ModelArchive ID ma-9gsmk. Chlamydomonas reinhardtii TRAP-Sec61-RAMP4-eL38 complex. https://modelarchive.org/doi/10.5452/ma-9gsmk

Lewis AJO Hegde RS (2024) ModelArchive ID ma-l9qqq. Saccharomyces cerevisiae Sec61-TRAP complex (aka Irc22p). https://modelarchive.org/doi/10.5452/ma-l9qqq

Lewis AJO Hegde RS (2024) ModelArchive ID ma-ise4t. Canis lupus familiaris BOS complex (TMEM147/Nicalin/NOMO, C. lupus familiaris). https://modelarchive.org/doi/10.5452/ma-ise4t

The following previously published data sets were used:

Tesina P Ebine S Buschauer R Thoms M Matsuo Y Inada T Beckmann R (2023) RCSB Protein Data Bank ID 8AGX. Molecular basis of eIF5A-dependent CAT tailing in eukaryotic ribosome-associated quality control. https://www.rcsb.org/structure/8AGX

Smirnova J Loerke J Kleinau G Schmidt A Bürger J Meyer EH Mielke T Scheerer P Bock R Spahn CMT Zoschke R (2023) RCSB Protein Data Bank ID 8B2L. Structure of the actively translating plant 80S ribosome at 2.2 Å resolution. https://www.rcsb.org/structure/8B2L

Braunger K Pfeffer S Shrimal S Gilmore R Berninghausen O Mandon EC Becker T Förster F Beckmann R (2018) RCSB Protein Data Bank ID 6FTI. Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum. https://www.rcsb.org/structure/6FTI

Holm M Natchiar SK Rundlet EJ Myasnikov AG Watson ZL Altman RB Wang HY Taunton J Blanchard SC (2023) RCSB Protein Data Bank ID 8GLP. mRNA decoding in human is kinetically and structurally distinct from bacteria. https://www.rcsb.org/structure/8GLP

Holm M Natchiar SK Rundlet EJ Myasnikov AG Watson ZL Altman RB Wang HY Taunton J Blanchard SC (2023) EMDB ID EMD-40205. mRNA decoding in human is kinetically and structurally distinct from bacteria. https://www.ebi.ac.uk/emdb/EMD-40205

Pfeffer S Dudek J Schaffer M Ng BG Albert S Plitzko JM Baumeister W Zimmermann R Freeze HH Engel BD Forster F (2017) EMDB ID EMD-4145. Dissecting the molecular organization of the translocon-associated protein complex. https://www.ebi.ac.uk/emdb/EMD-4145

Braunger K Pfeffer S Shrimal S Gilmore R Berninghausen O Mandon EC Becker T Forster F Beckmann R (2018) EMDB ID EMD-4311. Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum. https://www.ebi.ac.uk/emdb/EMD-4311

Voorhees RM Hegde RS (2016) RCSB Protein Data Bank ID 3JC2. The structure of the mammalian Sec61 channel opened by a signal sequence. https://www.rcsb.org/structure/3JC2

Voorhees RM Hegde RS (2016) EMDB ID EMD-3245. The structure of the mammalian Sec61 channel opened by a signal sequence. https://www.ebi.ac.uk/emdb/EMD-3245

Smalinskaitė L Kim MK Lewis AJO Keenan RJHegde RS (2022) RCSB Protein Data Bank ID 7TM3. Mechanism of an intramembrane chaperone for multipass membrane proteins. https://www.rcsb.org/structure/7TM3

Smalinskaite L Kim MK Lewis AJO Keenan RJHegde RS (2022) EMDB ID EMD-25994. Mechanism of an intramembrane chaperone for multipass membrane proteins. https://www.ebi.ac.uk/emdb/EMD-25994

Gemmer M Chaillet M Lvan Loenhout J (2023) EMDB ID EMD-15885. Visualization of translation and protein biogenesis at the ER membrane. https://www.ebi.ac.uk/emdb/EMD-15885

Gemmer M Chaillet M Lvan Loenhout J (2023) EMDB ID EMD-15889. Visualization of translation and protein biogenesis at the ER membrane. https://www.ebi.ac.uk/emdb/EMD-15889

Gemmer M Fedry JMM Forster FG (2022) RCSB Protein Data Bank ID 8B6L. Subtomogram average of the human Sec61-TRAP-OSTA-translocon. https://www.rcsb.org/structure/8B6L

Gemmer M Fedry JMM Forster FG (2022) EMDB ID EMD-15870. Subtomogram average of the human Sec61-TRAP-OSTA-translocon. https://www.ebi.ac.uk/emdb/EMD-15870

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Additional details

Identifiers

DOI
10.7554/eLife.95814.3
Other
oai:uchicago.tind.io:12927

Funding

Medical Research Council
United Kingdom Research and Innovation
National Institutes of Health
R35 GM145374
National Institutes of Health
T32 GM007183

UChicago Information

Division(s)
Biological Sciences Division
Department(s)
Biochemistry and Molecular Biology, Molecular Genetics and Cell Biology