Extended Data Fig. 7: Analysis of the substrate-engaged multipass translocon.

a, RNCs of FLAG-TRAM2 truncated at the indicated lengths (see diagram) were synthesised in RRL supplemented with HEK293 SP cells. The RNCs were affinity-purified under native conditions via the N-terminal FLAG-tag (as in Extended Data Fig. 1f) and analysed by immunoblotting for the indicated proteins. The negative control (neg) is a translation reaction without mRNA. b, RNCs of Twin-Strep Tag (TST)-Rho^ext (see diagram) truncated 40 residues downstream of TMD2, TMD3, TMD5 or TMD7 were analysed for PAT complex association as in Fig. 1b. The RNC of 7+ length represents a nascent chain truncated 70 residues beyond TMD7. c, RNCs of FLAG-TRAM2 (see diagram) truncated 40 residues downstream of indicated TMD were analysed for PAT complex association as in Extended Data Fig. 1f. d, Photo-crosslinking analysis of ³⁵S-methionine-labelled Rho-4TMD translocation intermediate (as in Fig. 4d) via BPA installed at amber codons in TMD1 (Amb1), TMD3 (Amb3), or both. The top panel shows the autoradiograph of total RNCs after isolation by ultracentrifugation. Three of the key samples were also subjected to immunoprecipitation using the indicated antibodies and analysed in the bottom panel by autoradiography. Non-glycosylated and glycosylated substrate (Rho and Rho+glyc., respectively) and the adducts to Asterix (x-Asterix), Sec61β (x-S61β) and Sec61α (x-S61α) are indicated. e, Photo-crosslinking analysis of ³⁵S-methionine-labelled Rho-4TMD translocation intermediate via BPA installed at TMD2 (position 85). Three types of semi-permeabilised cells were compared: wild type (WT), TMCO1 KO (∆TMCO1) and ∆TMCO1 cells transiently transfected with FLAG-tagged TMCO1. The major crosslink seen with WT SP cells is lost in ∆TMCO1 SP cells, where other weak crosslinks to similar-sized unidentified proteins are seen. When a subset of the ∆TMCO1 cells now express FLAG-TMCO1, a new crosslink is seen that migrates slightly slower than the major product seen in WT cells. These results verify that the major crosslink in WT cells is TMCO1 and the slightly larger crosslink in the reconstituted ∆TMCO1 cells is FLAG-TMCO1. Crosslinking with BPA at different positions in TMD2 show that its more hydrophilic face interacts with TMCO1 (summarised in the diagram). f, Protease protection assay of Rho-4TMD RNC in WT or ∆TMCO1 SP cells. The autoradiograph shows equal glycosylation indicative of equal efficiencies of TMD1 insertion, but different amounts of fully-protected product indicative of successful 3-TMD insertion. In ∆TMCO1 cells, a larger population of proteins failed to insert TMDs 2 and 3, leading to protease-accessibility (see diagram). PK digestion in the presence of detergent (subscripted d) leads to complete digestion. Graph shows quantification of three independent experiments showing the mean and standard deviation. The difference observed is statistically significant (p = 0.01) by the two-tailed Student’s t-test. The source data for this graph is in Supplementary Table 4. g, RNCs of Rho^ext stalled 70 residues beyond TMD1 were assembled in WT, ∆Asterix, ∆CCDC47, ∆TMCO1 and ∆TMEM147 SP cells and affinity-purified via the FLAG-tag under native conditions as in Fig. 1b. Total cell lysates and purified RNCs were analysed by immunoblotting for the indicated proteins. h, Bismaleimidohexane (BMH) mediated crosslinking via cysteine in place of F56 in TMD1 of Rhodopsin stalled 70 residues downstream of TMD1 (top panel), or the equivalent RNC in which TMD1 is replaced with 22 leucine residues and a similarly positioned cysteine residue (bottom panel). ³⁵S-methionine-labelled RNCs were assembled with WT, ∆Asterix, ∆TMCO1, and ∆TMEM147 SP cells, subjected to BMH crosslinking where indicated, and either directly analysed by SDS-PAGE and autoradiography or after denaturing anti-Asterix IP. Equal glycosylation efficiency in all samples without BMH crosslinking indicate TMD1 insertion is unaffected by the loss of MPT components. Asterix crosslinking (marked as x-Asx) is unimpaired in ∆TMCO1 and ∆TMEM147 SP cells. In ∆Asterix cells, TMD1 of Rho crosslinks to an unidentified product that migrates slightly faster than Asterix (upward arrowhead). The 22L TMD does not crosslink to Asterix or the unidentified product, suggesting these interactions need partial TMD hydrophilicity.