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 E3^UFM1-60S association. Representative immunoblot analysis of ribosome pellets or inputs from DDRGK1^KO 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 DDRGK1^KO 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-E3^UFM1 complexes sterically clash with the outer leaflet of the ER membrane. Cryo-EM maps for State 2 (e) and State 3 (f) 60S-UFM1-E3^UFM1 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 E3^UFM1 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 UFM1^KO 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.