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UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER

Nature volume 627, pages 445–452 (2024)Cite this article

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Abstract

Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour1. UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER)2,3. UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER3,4. Neither the molecular mechanism by which the UFMylation machinery achieves such precise target selection nor how this ribosomal modification promotes 60S rescue is known. Here we show that ribosome UFMylation in vivo occurs on free 60S and we present sequential cryo-electron microscopy snapshots of the heterotrimeric UFM1 E3 ligase (E3(UFM1)) engaging its substrate uL24. E3(UFM1) binds the L1 stalk, empty transfer RNA-binding sites and the peptidyl transferase centre through carboxy-terminal domains of UFL1, which results in uL24 modification more than 150 Å away. After catalysing UFM1 transfer, E3(UFM1) remains stably bound to its product, UFMylated 60S, forming a C-shaped clamp that extends all the way around the 60S from the transfer RNA-binding sites to the polypeptide tunnel exit. Our structural and biochemical analyses suggest a role for E3(UFM1) in post-termination release and recycling of the large ribosomal subunit from the ER membrane.

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Fig. 1: E3(UFM1) forms a long-lived complex with UFMylated 60S.
Fig. 2: Structural analysis of the 60S–UFM1–E3(UFM1) complex.
Fig. 3: Organization of the E3(UFM1) complex bound to UFMylated 60S.
Fig. 4: Sequential E3(UFM1) binding and UFMylation of 60S promotes SEC61 displacement.
Fig. 5: DeUFMylation promotes the dissociation of 60S and E3(UFM1).

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Data availability

All relevant data are included in the manuscript and Supplementary Information. MS data files have been uploaded to the MassIVE proteomics database (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) with the identifiers MSV000093510 and MSV000093721 for mt-UFM1 proximity and SBP-UFM1 pulldown experiments, respectively. The cryo-EM structural data generated in this study have been deposited in the Electron Microscopy Data Bank under the following accession codes: EMD-16903 for the 60S–UFM1–E3(UFM1) (obtained from the UFM1 pull-down); EMD-16908 for the 60S–E3(UFM1)–SEC61 complex in state 1 (PDB accession 8OJ8); EMD-16902 for the 60S–UFM1–E3(UFM1)–SEC61 complex in state 2 (PDB accession 8OJ0); EMD-16880 for the 60S–UFM1–E3(UFM1) complex in state 3 (states 1, 2 and 3 obtained from the UFL1 pull-down) (PDB accession 8OHD); and EMD-16905 for the in vitro reconstituted 60S–UFM1–E3(UFM1) complex (state 3 (PDB accession 8OJ5). The structures used for atomic model building of 60S–UFM1–E3(UFM1) and 60S–UFM1–E3(UFM1)–SEC61 complexes are available from Worldwide Protein Data Bank (wwPDB) with accession codes 8GLP, 8G5Y, 6Z6M and 6W6L.

References

  1. Cappadocia, L. & Lima, C. D. Ubiquitin-like protein conjugation: structures, chemistry, and mechanism. Chem. Rev. 118, 889–918 (2018).

    Article  CAS  PubMed  Google Scholar 

  2. Walczak, C. P. et al. Ribosomal protein RPL26 is the principal target of UFMylation. Proc. Natl Acad. Sci. USA 116, 1299–1308 (2019).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  3. Wang, L. et al. UFMylation of RPL26 links translocation-associated quality control to endoplasmic reticulum protein homeostasis. Cell Res. 30, 5–20 (2020).

    Article  CAS  PubMed  Google Scholar 

  4. Scavone, F., Gumbin, S. C., DaRosa, P. A. & Kopito, R. R. RPL26/uL24 UFMylation is essential for ribosome-associated quality control at the endoplasmic reticulum. Proc. Natl Acad. Sci. USA 120, e2220340120 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Millrine, D., Peter, J. J. & Kulathu, Y. A guide to UFMylation, an emerging posttranslational modification. FEBS J. 290, 5040–5056 (2023).

    Article  CAS  PubMed  Google Scholar 

  6. Peter, J. J. et al. A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation. EMBO J. 41, e111015 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ishimura, R. et al. Mechanistic insights into the roles of the UFM1 E3 ligase complex in ufmylation and ribosome-associated protein quality control. Sci. Adv. 9, eadh3635 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Gerakis, Y., Quintero, M., Li, H. & Hetz, C. The UFMylation system in proteostasis and beyond. Trends Cell Biol. 29, 974–986 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Xie, Z., Fang, Z. & Pan, Z. Ufl1/RCAD, a Ufm1 E3 ligase, has an intricate connection with ER stress. Int. J. Biol. Macromol. 135, 760–767 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat. Biotechnol. 36, 880–887 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kang, S. H. et al. Two novel ubiquitin-fold modifier 1 (Ufm1)-specific proteases, UfSP1 and UfSP2. J. Biol. Chem. 282, 5256–5262 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Millrine, D. et al. Human UFSP1 is an active protease that regulates UFM1 maturation and UFMylation. Cell Rep. 40, 111168 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Russell, D. W. & Spremulli, L. L. Mechanism of action of the wheat germ ribosome dissociation factor: interaction with the 60S subunit. Arch. Biochem. Biophys. 201, 518–526 (1980).

    Article  CAS  PubMed  Google Scholar 

  14. Gartmann, M. et al. Mechanism of eIF6-mediated inhibition of ribosomal subunit joining. J. Biol. Chem. 285, 14848–14851 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lyumkis, D. et al. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex. Proc. Natl Acad. Sci. USA 111, 15981–15986 (2014).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  16. Shao, S., Brown, A., Santhanam, B. & Hegde, R. S. Structure and assembly pathway of the ribosome quality control complex. Mol. Cell 57, 433–444 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Shen, P. S. et al. Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Science 347, 75–78 (2015).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  18. Jumper, J. & Hassabis, D. Protein structure predictions to atomic accuracy with AlphaFold. Nat. Methods 19, 11–12 (2022).

    Article  CAS  PubMed  Google Scholar 

  19. Evans, R. et al. Protein complex prediction with AlphaFold-Multimer. Preprint at bioRxiv https://doi.org/10.1101/2021.10.04.463034 (2022).

  20. Halic, M. et al. Signal recognition particle receptor exposes the ribosomal translocon binding site. Science 312, 745–747 (2006).

    Article  CAS  PubMed  ADS  Google Scholar 

  21. Beckmann, R. et al. Alignment of conduits for the nascent polypeptide chain in the ribosome–Sec61 complex. Science 278, 2123–2126 (1997).

    Article  CAS  PubMed  ADS  Google Scholar 

  22. Habisov, S. et al. Structural and functional analysis of a novel interaction motif within UFM1-activating enzyme 5 (UBA5) required for binding to ubiquitin-like proteins and ufmylation. J. Biol. Chem. 291, 9025–9041 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Padala, P. et al. Novel insights into the interaction of UBA5 with UFM1 via a UFM1-interacting sequence. Sci. Rep. https://doi.org/10.1038/s41598-017-00610-0 (2017).

  24. Martinez-Sanchez, A. et al. Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms. Nat. Methods 17, 209–216 (2020).

    Article  CAS  PubMed  Google Scholar 

  25. Braunger, K. et al. Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum. Science 360, 215–219 (2018).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  26. McGilvray, P. T. et al. An ER translocon for multi-pass membrane protein biogenesis. eLife 9, e56889 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Smalinskaitė, L., Kim, M. K., Lewis, A. J. O., Keenan, R. J. & Hegde, R. S. Mechanism of an intramembrane chaperone for multipass membrane proteins. Nature 611, 161–166 (2022).

    Article  PubMed  PubMed Central  ADS  Google Scholar 

  28. Sundaram, A. et al. Substrate-driven assembly of a translocon for multipass membrane proteins. Nature 611, 167–172 (2022).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  29. Blobel, G. Extraction from free ribosomes of a factor mediating ribosome detachment from rough microsomes. Biochem. Biophys. Res. Commun. 68, 1–7 (1976).

    Article  CAS  PubMed  ADS  Google Scholar 

  30. Borgese, D., Blobel, G. & Sabatini, D. D. In vitro exchange of ribosomal subunits between free and membrane-bound ribosomes. J. Mol. Biol. 74, 415–438 (1973).

    Article  CAS  PubMed  Google Scholar 

  31. Grau-Bové, X., Sebé-Pedrós, A. & Ruiz-Trillo, I. The eukaryotic ancestor had a complex ubiquitin signaling system of archaeal origin. Mol. Biol. Evol. 32, 726–739 (2015).

    Article  PubMed  Google Scholar 

  32. Picchianti, L. et al. Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy. EMBO J. 42, e112053 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jan, C. H., Williams, C. C. & Weissman, J. S. Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling. Science 346, 1257521 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Jaako, P. et al. eIF6 rebinding dynamically couples ribosome maturation and translation. Nat. Commun. 13, 1562 (2022).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  35. Weis, F. et al. Mechanism of eIF6 release from the nascent 60S ribosomal subunit. Nat. Struct. Mol. Biol. 22, 914–919 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Leto, D. E. et al. Genome-wide CRISPR analysis identifies substrate-specific conjugation modules in ER-associated degradation. Mol. Cell 73, 377–389.e11 (2019).

    Article  CAS  PubMed  Google Scholar 

  37. An, H., Ordureau, A., Körner, M., Paulo, J. A. & Harper, J. W. Systematic quantitative analysis of ribosome inventory during nutrient stress. Nature 583, 303–309 (2020).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  38. McAlister, G. C. et al. Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses. Anal. Chem. 84, 7469–7478 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Paulo, J. A., O’Connell, J. D. & Gygi, S. P. A triple knockout (TKO) proteomics standard for diagnosing ion interference in isobaric labeling experiments. J. Am. Soc. Mass. Spectrom. 27, 1620–1625 (2016).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  40. Schweppe, D. K. et al. Characterization and optimization of multiplexed quantitative analyses using high-field asymmetric-waveform ion mobility mass spectrometry. Anal. Chem. 91, 4010–4016 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Erickson, B. K. et al. Active instrument engagement combined with a real-time database search for improved performance of sample multiplexing workflows. J. Proteome Res. 18, 1299–1306 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Schweppe, D. K. et al. Full-featured, real-time database searching platform enables fast and accurate multiplexed quantitative proteomics. J. Proteome Res. 19, 2026–2034 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Elias, J. E. & Gygi, S. P. Target–decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Elias, J. E. & Gygi, S. P. Target–decoy search strategy for mass spectrometry-based proteomics. Methods Mol. Biol. https://doi.org/10.1007/978-1-60761-444-9_5 (2010).

  45. Huttlin, E. L. et al. A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–1189 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).

    Article  CAS  PubMed  Google Scholar 

  47. Itzhak, D. N., Tyanova, S., Cox, J. & Borner, G. H. Global, quantitative and dynamic mapping of protein subcellular localization. eLife 5, e16950 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Ishihama, Y., Rappsilber, J. & Mann, M. Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics. J. Proteome Res. 5, 988–994 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Ha, B. H. et al. Structural basis for Ufm1 processing by UfSP1. J. Biol. Chem. 283, 14893–14900 (2008).

    Article  CAS  PubMed  Google Scholar 

  50. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  53. Wagner, T. et al. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun Biol. 2, 218 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  55. Wells, J. N. et al. Structure and function of yeast Lso2 and human CCDC124 bound to hibernating ribosomes. PLoS Biol. 18, e3000780 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Kimanius, D., Dong, L., Sharov, G., Nakane, T. & Scheres, S. H. W. New tools for automated cryo-EM single-particle analysis in RELION-4.0. Biochem. J. 478, 4169–4185 (2021).

    Article  CAS  PubMed  Google Scholar 

  58. Liang, X. et al. Structural snapshots of human pre-60S ribosomal particles before and after nuclear export. Nat. Commun. 11, 3542 (2020).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  59. Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun. Biol. 4, 874 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  61. Holm, M. et al. mRNA decoding in human is kinetically and structurally distinct from bacteria. Nature 617, 200–207 (2023).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  62. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  ADS  Google Scholar 

  63. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  64. Yamashita, K., Palmer, C. M., Burnley, T. & Murshudov, G. N. Cryo-EM single-particle structure refinement and map calculation using Servalcat. Acta Crystallogr. D Struct. Biol. 77, 1282–1291 (2021).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  65. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D Struct. Biol. 74, 519–530 (2018).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  66. Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    Article  CAS  PubMed  Google Scholar 

  67. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  CAS  PubMed  Google Scholar 

  68. Amici, D. R. et al. FIREWORKS: a bottom-up approach to integrative coessentiality network analysis. Life Sci. Alliance 4, e202000882 (2021).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank C. Ungewickell, S. Rieder and A. Gilmozzi for technical assistance; M. C. Riepe for discussion throughout this work and for critical reading of the manuscript; A. Ting for reagents and advice with the proximity labelling experiments; and Y. Liu from S2C2 for support and assistance. This study was supported by grants from the ERC (RELYUBL, 677623), the Lister Institute of Preventive Medicine, the BBSRC (BB/T008172/1) and the MRC (grant MC_UU_00018/3) to Y. K.; from the ERC (ADG 885711 Human-Ribogenesis and DFG (SFB/TRR-174, BE1814/15-1, BE1814/1-1) to R.B.; and from the NIH to R.R.K. (1R01GM148477 and 5R01GM074874), J.W.H. (RO1AG011085 and R01NS083524) and J.A.P. (K01DK098285). P.A.D. was supported by NIH training grant 5T32NS007280. S.C.G. was supported by NIH training grant T32GM007276 and by a Stanford Graduate Fellowship. Some of this work was performed at the S2C2, which is supported by the National Institutes of Health Common Fund Transformative High-Resolution Cryo-Electron Microscopy program (U24 GM129541). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Author notes

  1. Alban Ordureau

    Present address: Cell Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

  2. These authors contributed equally: Paul A. DaRosa, Ivan Penchev

Authors and Affiliations

  1. Department of Biology, Stanford University, Stanford, CA, USA

    Paul A. DaRosa, Samantha C. Gumbin, Francesco Scavone, Magda Wąchalska & Ron R. Kopito

  2. Department of Biochemistry, Gene Center, University of Munich, Munich, Germany

    Ivan Penchev, Thomas Becker & Roland Beckmann

  3. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Joao A. Paulo, Alban Ordureau & J. Wade Harper

  4. Medical Research Council Protein Phosphorylation and Ubiquitylation Unit (MRC-PPU), School of Life Sciences, University of Dundee, Dundee, UK

    Joshua J. Peter & Yogesh Kulathu

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Contributions

P.A.D., I.P., R.R.K., T.B. and R.B. conceived and designed the project. P.A.D. devised and performed all cell-based and cell-free biochemical experiments, except the translocon release assays, which were designed and performed by S.C.G. and M.W., and the AP accumulation assay, performed by F.S. P.A.D. prepared the cryo-EM grids for the in vitro UFMylated 60S samples. P.A.D., A.O., J.A.P. and J.W.H. conceived and designed the proximity labelling and affinity capture experiments. A.O. and J.A.P. performed the MS experiments. J.J.P. and Y.K. provided purified UFMylation enzymes and assisted with in vitro UFMylation. I.P. generated Flag–UFM1 and Flag–UFL1 constructs and cell lines, performed immunoisolation and processed all the cryo-EM data. P.A.D. and I.P. conducted and analysed the AlphaFold modelling. I.P. generated molecular models and, together with T.B. and R.B., analysed the E3(UFM1) structures. P.A.D., I.P., R.R.K., T.B. and R.B. wrote and edited the manuscript with input from all authors.

Corresponding authors

Correspondence to Roland Beckmann or Ron R. Kopito.

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Extended data figures and tables

Extended Data Fig. 1 E3UFM1 selectively modifies and then binds 60S ribosomes.

a, Workflow for UFM1 miniTurbo proximity profiling. b, Covalent modification of uL24 UFM1 by mT-UFM1 depends on expression of UBA5 and is enhanced by disruption of UFSP2. Immunoblot analysis of mT-UFM1 knock-in cell lines in the indicated genetic backgrounds used in the proximity labeling experiment in (a) and Fig. 1b. Note presence of non-specific band just above uL24-UFM1 band visible in UBA5KO. c, mT-UFM1 is conjugated to uL24 on ribosomes (control for experiment in a and Fig. 1b). Lysates of U2OS mT-UFM1 knock-in cells treated with or without 200 nM anisomycin for 20 min to induce ribosome collisions were analyzed before (input) or after pelleting (ribosome pellet) through a sucrose cushion. d, Proximity labeling with mT-UFM1 shows conjugation-dependent biotinylation of proteins. Time course of UFC1 and DDRGK1 biotinylation in U2OS mT-UFM1 knock-in cells in wildtype or UBA5KO background (as indicated) showing the conjugation-dependent specificity of biotin labeling. Cells were incubated with biotin for the indicated times prior to lysis, followed by streptavidin pulldowns (for biotinylated proteins), and elution from streptavidin beads by boiling in Laemmli buffer for immunoblot analysis. Based on the continued high selectivity for UFC1 biotinylation over the time course, mass spectrometry analysis was performed with a 4 h incubation with biotin (see Methods). e, Representative elutions from pull-downs as in Fig. 1b, c staining nitrocellulose with total protein stain (LI-COR Revert) or immunoblotted for uL24 to show the capture of ribosomes and enrichment of UFMylated uL24 (~80 % UFMylated). Transiently expressed eL36-SBP used to isolate ribosomes results in characteristic ribosome band patterning seen in SBP-UFM1 pulldowns, but lack bands (black arrowheads) discernable in the SBP-UFM1 pulldown that likely correspond to UFL1 and DDRGK1 (by molecular weight). Untagged UFM1 is used as a negative control. f, Sucrose density sedimentation profile for experiment in (g). g, UFM1 modifies exclusively 60S in vivo. Lysates from wildtype K562 cells were fractionated on sucrose density gradients and analyzed by immunoblot with the indicated antibodies. Sedimentation behavior of UFMylated uL24 parallels that of the obligatory 60S markers NEMF and eIF6. h, Quantification of indicated bands for fractions in (g) showing correlations between UFMylated uL24 and NEMF (upper) and eIF6 (lower). i, Validation of cell lines (lanes 7–9) and UFM1 and UFSP2 distribution in fractions (lanes 1–6) used for the sucrose density sedimentation in Fig. 1d. Clonal K562 cell knockouts of UFSP2 and UFM1 show no detectable expression of UFSP2 and UFM1, respectively. Cell lysates were separated via sequential detergent fractionation into, cytosolic (“C”), and membrane (“M”) fractions and analyzed by immunoblot with indicated antibodies. Non-fractionated whole cell lysate, “WC”. This fractionation distinguishes the cytosolic UFC1-UFM1 adduct (an isopeptide linked conjugate) from the co-migrating uL24-UFM1 conjugate as reported previously2,6,12. j, Additional fractionation controls as in (i) for samples used in Fig. 1d showing partitioning of ER membrane and cytosolic markers. Membrane fractions are highly enriched for membrane markers (DDRGK1 and SEC61β) and lack cytosolic contaminants (e.g., GAPDH). k, Ribosome collisions increase E3UFM1–60S association. K562 cells were treated with or without 200 nM anisomycin (ANS) for 1 h to induce ribosome collisions. Lysates were sedimented through 1 M sucrose to isolate ribosomes and analyzed by immunoblotting with the indicated antibodies. l, Quantification of mono-UFMylated uL24, UFL1, DDRGK1, and CDK5RAP3 from biological triplicates in experiment as in (k). Data show mean ± SD for n = 3 biological replicates. m, 60S ribosomes are the preferred target of UFMylation in vitro. Sucrose density sedimentation analysis of in vitro UFMylation reaction containing a 1:2 60S:80S molar ratio showing selectivity for 60S ribosome modification. n, 80S ribosomes are poor substrates of UFMylation in vitro. Sucrose density sedimentation as in (m) with the same concentration of 80S ribosomes as substrate showing strongly reduced UFMylation and E3UFM1 binding. Source data is available in Supplementary Fig. 5 (for b-e and g-n), Supplementary Table 3 (for h), and Supplementary Table 4 (for l). Data in b-e, g, k, m, and n were replicated at least twice with similar results; for detailed descriptions see “Statistics and reproducibility” section of the Methods. The mobility of molecular weight markers (in kDa) is indicated on the left hand side of the blots in panels b-e, g, i-k, m, n.

Extended Data Fig. 2 Coessential relationship between UFMylation and 60S recycling pathway genes.

a, The UFMylation pathway exhibits strong co-essentiality with genes involved in 60S ribosome biogenesis (green circles) and N-glycosylation genes (blue circles). All UFMylation pathway genes (yellow circles) were used as input for the FIREWORKS (https://mendillolab.shinyapps.io/fireworks/)68. b, Table of DEPMAP co-dependencies for UFM1 network showing strong Pearson correlations with UFM1 pathway (yellow) and with 60S biogenesis factors EFL1, SBDS, and DNAJC21 (green). c, The UFL1-CTD binding site is incompatible with EFL1 and SBDS binding. Overlay of 60S-UFM1-E3UFM1 complex in State 3 with 60S-bound SBDS and EFL1 before displacement of eIF6 (PDB 5ANB)35. Note that the CTD of UFL1 (orange) would sterically clash with SBDS and EFL1, suggesting that eIF6 eviction may not occur until E3UFM1 has dissociated.

Extended Data Fig. 3 Cryo-EM data analysis and classification of native and reconstituted 60S-UFM1-E3UFM1 complexes.

a, Data processing scheme for the native 60S-UFM1-E3UFM1 complex derived from a FLAG-UFM1 pulldown. 616,046 particles were picked from 11,658 micrographs using crYOLO. Following 2D classification in cryoSPARC, 83,447 particles corresponding to 60S ribosomal subunits were selected. A consensus refinement was performed followed by CTF refinement in RELION. The 60S particles were then subjected to several rounds of 3D classification using a soft mask focusing on regions where non-ribosomal extra density was observed. This revealed one stable class consisting of 14,144 particles (16,9% of all 60S particles) that was refined to an average resolution of 3.1 Å. b, Data processing scheme for the in vitro reconstituted 60S-UFM1-E3UFM1 complex. 1,136,353 particles were picked from 10,692 micrographs using RELION’s AutoPick LoG algorithm. 2D classification revealed 846,919 ribosomal particles. 3D classification with a focused mask around the 40S revealed a number of classes, a majority of which were 60S particles with and without a bound E-site tRNA. In addition, two classes representing empty 80S ribosomes were found. Using a soft mask focusing on regions where non-ribosomal extra density was observed, one stable class consisting of 35,935 particles (4.6% of all particles) was isolated representing the stable 60S-UFM1-E3UFM1 complex. This class was refined to an average resolution of 2.9 Å.

Extended Data Fig. 4 Overall and local resolution and angular distribution of 60S-UFM1-E3UFM1 complexes.

(Local) resolution and angular distribution was assessed in RELION for the FLAG-UFM1 pulldown and the in vitro reconstituted sample (both resulting in State 3; see Extended Data Fig. 3) and the FLAG-UFL1 pulldown sample (yielding states 1, 2 and 3; see Extended Data Fig. 7). a, Gold-standard Fourier Shell Correlation (FSC) curves (obtained from RELION) for the reconstructions of the three states obtained from the UFL1 pulldown sample (states 1–3), the in vitro reconstituted sample (state 3) and the UFM1-pulldown sample (also state 3). CC = correlation corrected; UM = unmasked maps; MM = masked maps; PRMM = phase randomized masked maps. b, Cryo-EM maps, displayed after Gaussian low-pass filtering at a standard deviation of 2 in ChimeraX and colored according to local resolution. Shown are the entire 60S-UFM1-E3UFM1 reconstructions (60S) as well as isolated densities for the E3UFM1 (E3) and the SEC61 complex (SEC61; visualized in states 1 and 2 as obtained from the FLAG-UFL1 pulldown sample). c, Original (unfiltered) cryo-EM maps and angular distribution plots for final 60S-UFM1-E3UFM1 complex reconstructions obtained from RELION. The height and color (from blue to red) of the cylinder bars is proportional to the number of particles in those views.

Extended Data Fig. 5 Models and mutational analyses of the 60S-UFM1-E3UFM1 complex.

a, Final model of the UFM1-E3UFM1 complex derived from fitting the AlphaFold model (shown in (b) into the cryo-EM map of the reconstituted 60S-UFM1-E3UFM1 complex. This conformation represents State 3. b, AlphaFold model of the UFM1-E3UFM1 complex. In this model, the C-terminal region (480–794) of UFL1 was derived from AlphaFold 2 prediction18 and an N-terminal fragment (1–389) was used for AlphaFold-Multimer modeling19. The models are colored according to a per-model confidence score (pLDDT; from 0 to 100). Blue regions display a very high confidence (pLDDT > 90), red region low confidence (pLDDT <50). c, Overlay of the initial AlphaFold model (green) with the final cryo-EM model (grey). d, Multiple sequence alignment of the DDRGK1 UFIM and flanking regions. Intensity of color (blue/violet) represents the % sequence identity. Mutated residues in UFIMmt are indicated by asterisks. Conserved residues that contact UFM1 at the C-terminal end of the DDRGK1 exit-binding helix (EBH; shown in (e) and (f) are highlighted in red. e, Close-up of the DDRGK1 UFIM and EBH interactions with UFM1 derived from AlphaFold-Multimer prediction and consistent with cryo-EM density map. Side chain interactions predicted by AlphaFold-Multimer at this interface are noted. f, Same as (e), but with the amino acids that were mutated in Figs. 4d, h and i within the UFIM highlighted in purple. g, Crystal structure of the UFIM of UBA5 in complex with UFM1 (PDB 5IA8)23. Like the DDRGK1 UFIM, the UBA5 UFIM also establishes a β-augmentation with β2 of UFM1, however, neither the sequence nor the overall conformation of these UFIMs are conserved. h, Molecular model and schematic representation of the DDRGK1 ΔEBM mutant used in Figs. 4d, h and i. The truncated regions are depicted in gray. i, Molecular model and schematic representation of UFL1 C-terminal deletion mutants used in Fig. 4c. Truncated regions are depicted in gray. UFL1(1–532) = ΔCTD.

Extended Data Fig. 6 Map quality, model fitting and molecular interactions of 60S-UFM1-E3UFM1 complexes.

Shown are fits of the E3UFM1 complex model (in ribbons) into cryo-EM maps (transparent surface) of the FLAG-UFL1 native pulldown sample (States 1–3; native) and the in vitro reconstituted sample (state 3; in vitro). a, Views highlighting the interactions of the UFL1 C-terminal regions (UFL1-C) with the 60S. The maps are shown unmodified after refinement (upper row) or low-pass filtered at 5 Å (center and bottom rows) to visualize more flexible parts. Bottom row, close-up of the UFL1-C in center row; alternate angle. UFL1, yellow; 60S, grey. b, Views highlighting the interaction network of CDK5RAP3 and DDRGK1 with the UFL1 scaffold. Upper row; central region of the E3 complex (E3) with multiple interactions between UFL1 (yellow) and CDK3RAP5 (blue) near uL13. Lower row; UFL1/DDRGK1(magenta) interface (pHW complementation). In state 1, these parts of the complex are not resolved. c, Views focusing on the DDRGK1 EBH (upper row) and close-up view on the DDRGK1 region near uL24-conjugated UFM1 (lower row; uL24, light pink; UFM1, green). Here, β-augmentation is predicted by AlphaFold formed by UFM1 and the UFIM-containing linker region between the DDRGK1 EBH and the DDRGK1 WH. The cryo-EM maps were low-pass filtered at 5 Å and show experimental evidence for predicted β-augmentation. Note that in state 1, these parts of the complex are not visualized and in states 1 and 2, the DDRGK1 EBH is not positioned. d, Views focusing on the structured PTC loop region (D416-V448) of the UFL1 disordered domain (N391-F479) identified near the peptidyl transferase center (PTC) of the 60S. In the best resolved density map for this region (State 3 from FLAG-UFL1 pulldown), a clear helical density is present that fits the α-helical part of the N-terminus of this region (State 3, upper right). We clearly observe densities for three basic residues (K417, R422 and R423); UFL1 Y443 engages in stacking interactions with 25S rRNA base A4548 (lower right; see also Fig. 3f).

Extended Data Fig. 7 Cryo-EM data analysis and classification of native 60S-E3UFM1 complexes derived from the FLAG-UFL1 pulldown.

From 50,993 micrographs, 3,017,721 particles were picked using RELION AutoPick and used for 2D classification, which yielded a total of 1,247,589 ribosomal particles. 3D classification with a mask focusing around the 40S revealed 132,004 inactive 80S ribosomes harboring eEF2 and EBP1 (ref. 55), with the remainder of particles being 60S subunits. The 60S particles were further classified with a mask focusing around the A- and P- sites of the 60S, revealing 37,714 particles corresponding to a biogenesis intermediate featuring LSG1, NMD3, and ZNF622 (PDB 6LSR58). No density corresponding to the UFMylation machinery was found in this class. The remaining particles were sorted with a mask focusing around the tunnel exit of the 60S, revealing three major subsets. One subset featured the DDRGK1 EBH and was further classified, revealing the entire E3 complex at a final resolution of 3 Å. This state was dubbed state 3. The second subset featured SEC61 bound to the 60S, and downstream classification revealed two distinct classes, one with the entire E3 bound, but with a delocalized DDRGK1 helix, and a second featuring only the C-terminal region. These states were refined to final resolutions of 3.33 and 3.27 Å, respectively, and were dubbed States 2 and 1. The last subset featured either EBP1 or no density around the tunnel exit, and further classification showed that these were empty 60S subunits.

Extended Data Fig. 8 SEC61 model fitting in States 1 and 2.

a, Three views of the SEC61 complex density from State1, low-pass filtered at 5 Å (transparent grey) with fitted model of SEC61 in closed state (PDB 6W6L)26. Left and center views at the C-terminal half of SEC61α including the ribosome anchor (loops 6-7 and 8-9) and the amphipathic helix of SEC61γ. This region is usually well-resolved in ribosome-SEC61 cryo-EM reconstructions and we observe helical density for transmembrane helices 5–9 of SEC61α and the N-terminal amphipathic helix of SEC61γ. Right; View at the N-terminal half of SEC61α and SEC61β. Here, the density is expectedly rather weak and only visible at very low contour levels. The N-terminal half is more flexible, especially in case the complex is not engaged with a nascent peptide substrate. b, c, Close-up views at the ribosome binding site of SEC61α, consisting of loops L6-7 and L8-9 (unfiltered density map). Here, density for these loops could be unambiguously fitted. d-f, same as (a-c), but for state 2.

Extended Data Fig. 9 UFMylation of 60S promotes SEC61 displacement.

a, b, Two views showing steric clashes between the N-terminal tip of the DDRGK1 EBH with SEC61 at the 60S tunnel exit site. Shown is an overlay of the DDRGK1 helix from State 3 (pink) with SEC61 from State 2 (model shown as transparent surface; SEC61α, light green; SEC61γ light blue). c, Mutation of the DDRGK1 UFIM reduces ribosome E3UFM1-60S association. Representative immunoblot analysis of ribosome pellets or inputs from DDRGK1KO HEK293 cells transiently replaced with indicated DDRGK1 variants. UFMylation was stimulated with anisomycin to enhance the detection of the low abundance E3-ribosome association. d, Quantification of UFL1 and CDK5RAP3 band intensities of ribosome pellets as in (c) from biological triplicates. Data show mean ± SD relative to DDRGK1KO HEK293 rescued with WT DDRGK1. P values in plots for the indicated comparisons were derived from one-way ANOVA and Dunnett’s multiple comparison tests for n = 3 biological replicates. e, f, 60S-UFM1-E3UFM1 complexes sterically clash with the outer leaflet of the ER membrane. Cryo-EM maps for State 2 (e) and State 3 (f) 60S-UFM1-E3UFM1 complexes were fitted into cryo-ET maps of mammalian ER-membrane-bound 80S ribosomes (EMD-0084)24 to obtain an outline of the lipid bilayer (gray dashed lines). The observed position of UFM1 and the bound E3UFM1 would partially clash with the ER membrane in State 2 requiring a slight tilt of the ribosome at the SEC61-ribosome junction to accommodate stable E3 association. In State 3, the DDRGK1 EBH would reach deep into the lipid bilayer and could only be accommodated with a substantial tilt or full dissociation of the ribosome from the SEC61 complex. g, Quantification of SEC61α band intensities in ribosome pellets, as in Fig. 4e. Data show mean ± SD relative to untreated and p values from indicated comparisons derived from a two-way ANOVA followed by uncorrected Fisher’s LSD for n = 2 biological replicates. h, Quantification of SEC61α band intensities in ribosome pellets, as in 4f. Data show mean ± SD relative to untreated and p values from indicated comparisons derived from a two-way ANOVA followed by uncorrected Fisher’s LSD for n = 3 biological replicates. i, UFMylation is required for timely dissociation of 60S from translocon following translation termination. Immunoblot analysis of ribosome pellets or inputs from WT or UFM1KO HEK293 cells treated with 3.75 µM harringtonine for the indicated times. Quantification of SEC61α or SEC61β band intensities in ribosome pellets, as in 4e,f. Data show mean ± SD relative to untreated and p values from indicated comparisons derived from a two-way ANOVA followed by uncorrected Fisher’s LSD for n = 3 biological replicates. Source data is available in Supplementary Fig. 6 (for c-d and g-i) and Supplementary Tables 5, 6, 7 and 8 (for d, g, h and i, respectively). All experiments were replicated at least twice; for p values and detailed descriptions of data replications see “Statistics and reproducibility” section of the Methods. The mobility of molecular weight markers (in kDa) is indicated on the left hand side of the blots in panels c and i.

Extended Data Table 1 Cryo-EM data collection, refinement and model validation
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DaRosa, P.A., Penchev, I., Gumbin, S.C. et al. UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER. Nature 627, 445–452 (2024). https://doi.org/10.1038/s41586-024-07073-0

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