Fig. 4: Multipass-translocon-dependent topogenesis.

a, Top: YIPF1 harbours a single N-glycosylation site (black circle) near its C terminus. Bottom: [³⁵S]methionine-labelled wild-type and mutant (N297A) YIPF1 were translated in RRL in the presence of rough microsomes, isolated by sedimentation, and analysed by autoradiography. b, Flag–YIPF1 was translated in RRL with wild-type or TMCO1-knockout (ΔT) rough microsomes, isolated by sedimentation, and analysed either directly (input) or after alkaline sodium carbonate extraction. YIPF1, TMCO1, BIP (ER luminal) and TRAPα (ER integral) were visualized by immunoblotting. The proportion of glycosylated (Glyc.) YIPF1 is indicated. c, [³⁵S]methionine-labelled, C-terminally haemagglutinin (HA)-tagged YIPF1 was translated in RRL with wild-type or ΔT rough microsomes, isolated by sedimentation, and analysed by autoradiography before (−PK) or after (+PK) proteinase K treatment. The PK-treated sample was also analysed after immunoprecipitation using the HA tag. Full-length YIPF1–HA, its protease-protected fragments (PF), and the proportion of recovered PF are indicated. d, HA–YIPF1, HA–ASGR1 and TMED2–HA were translated in RRL with wild-type, single-knockout (TMCO1 (ΔT), Nicalin, (ΔN) or CCDC47 (ΔC)) or double-knockout (TMCO1/Nicalin (ΔTN) or TMCO1/CCDC47 (ΔTC)) rough microsomes, isolated by sedimentation, and analysed by immunoblotting. e, Quantification of YIPF1 glycosylation for n = 3 biological replicates, as in d. The data are shown as the mean ± s.d. f, Flag–YIPF1 was transiently transfected into wild-type or knockout cells, and total lysates were analysed by immunoblotting. g, Quantification of YIPF1 glycosylation as in f, for n = 5 biological replicates. The data are shown as the mean ± s.d. h, Reporter constructs to monitor protein stability in cells. i,j, Stably integrated HEK293 reporter lines were treated with the indicated short interfering RNA (siRNAs), induced with doxycycline, and analysed by flow cytometry. The histograms show FP ratios for each siRNA–reporter pair; the vertical black line indicates the mode of the control population. Statistical analyses in e,g were performed by ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (single pooled variance) in GraphPad Prism 9.4.0. **P < 0.0021; ***P < 0.0002; ****P < 0.0001; P values are given in Supplementary Table 1.