Fig 2: Translocon dynamics during TRAM2 synthesis.a, Template used to generate truncated, Flag-tagged TRAM2 constructs, as in Fig. 2a. b, Stalled, Flag-tagged TRAM2 constructs truncated at the indicated positions were analyzed as in Fig. 2b. Note that the 140-mer intermediate (and beyond) is glycosylated at position 55. c, Stalled, Flag-tagged TRAM2 constructs truncated at the indicated positions were translated in RRL in the presence of WT and double knockout (TMCO1/CCDC47, ‘ΔTC’) rough microsomes, and analyzed as in (b).

Fig 3: Natively isolated TMCO1-ribosome complexes contain multiple transmembrane components.(A) Emetine- and micrococcal nuclease-treated membranes from wild-type (WT) or 3xFlag-TMCO1 (Flag) HEK293 cells were digitonin-solubilized, immunoprecipitated via the 3xFlag tag on TMCO1, and the eluate sedimented through a sucrose cushion to isolate the ribosome-associated fraction for analysis. (B) Proteins enriched in the ribosomal fraction after immunoprecipitation from 3xFlag-TMCO1 or wild-type membranes. (C) Top hits were confirmed by western blotting. The catalytic STT3A subunit of the OST complex is not detected. (D) Topology and domain structure for the top hits, based on Uniprot annotation, homology modeling, de novo structure prediction (in RaptorX-Contact), and experimental mapping; the Sec61 complex is not shown. Distinguishing features include the large globular luminal domain of Nicalin (in contrast with the flexible luminal domains of NOMO and CCDC47), the large globular cytosolic domain of CCDC47 (with a conserved C-terminal coiled-coil), and a conserved cytosolic coiled-coil between the first two TMDs of TMCO1. TMEM147 is the core, multi-pass subunit of the Nicalin-TMEM147-NOMO complex Dettmer et al., 2010; note that the short extra-membrane loops of TMEM147 make it difficult to detect by mass spectrometry.

Fig 4: Additional characterization of the in vitro system and validation of siRNA knockdowns.a, HA-YIPF1 was translated in RRL in the presence of WT, single- (TMCO1, ΔT; Nicalin, ΔN; CCDC47, ΔC) or double-knockout (TMCO1/Nicalin, ΔTN; TMCO1/CCDC47, ΔTC) rough microsomes at different concentrations (determined by absorbance at 260 nm), isolated by sedimentation, and analyzed by SDS-PAGE and immunoblotting. The percentage of glycosylated YIPF1 is indicated below the gel. b, Plot of the data in (A). c, Membranes prepared from the indicated wild-type, single- or double-knockout HEK293 cell lines were analyzed by SDS-PAGE and immunoblotting. d, Stalled, Flag-tagged YIPF1 constructs truncated at position 183 and 277 were translated in RRL in the presence of wild-type (WT) or single knockout rough microsomes, and the membrane-associated fraction was isolated by sedimentation. Following anti-Flag immunoprecipitation of the digitonin-solubilized membranes, stalled RNCs were isolated by sedimentation and analyzed by SDS-PAGE and immunoblotting. e, Whole cell lysates from HEK293 cells treated with the indicated siRNAs were analyzed by SDS-PAGE and immunoblotting; ‘Ctrl’ is a non-targeting control siRNA.

Fig 5: Additional characterization of substrate features that direct assembly of the multipass translocon.a, The indicated ASGR1-derived intermediates were translated in RRL in the presence of wild-type HEK293 rough microsomes, and the membrane-associated fraction was isolated by sedimentation. Following anti-Flag immunoprecipitation of the digitonin-solubilized membranes, stalled RNCs were isolated by sedimentation and analyzed by SDS-PAGE and immunoblotting. b, Quantification of STT3A, TMCO1, CCDC47 and NOMO recruitment to stalled 2-TMD YIPF1 constructs as in Fig. 2f, for n = 5 biological replicates. Data are mean ± S.D. Analysis was performed using an RM one-way ANOVA with the Geisser-Greenhouse correction and Tukey’s multiple comparisons test as implemented in GraphPad Prism 9.4.0. *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; ****, p < 0.0001; p values are given in Supplementary Table 2.