Extended Data Fig. 1: Characterisation of the Rho^ext construct.

a, ³⁵S-methionine labelled Rho^ext ribosome nascent chain complexes (RNCs) of indicated length were synthesised in rabbit reticulocyte lysate (RRL) in the presence of canine pancreas-derived rough microsomes (RMs). Where indicated, RNCs were chemically crosslinked using bismaleimidohexane (BMH). The positions of the glycosylated (+glyc.) and non-glycosylated (-glyc.) translation products are indicated. The crosslink to Asterix (indicated by x-Asx) was verified by immunoprecipitation under denaturing conditions using anti-Asterix antibody (bottom panel). The pattern of PAT complex recruitment for Rho^extconstruct is similar to the non-extended Rho construct construct described previously¹⁰. b, ³⁵S-methionine labelled Rho^extRNC of the indicated length, lacking or containing the glycosylation site, was synthesised in RRL containing or lacking RMs. The translation products were then digested with proteinase K (PK). The population of polypeptides inserted into the membrane is protected from PK. c, Translation products were produced as in panel b (lanes 1 and 4), after which the RMs were isolated by sedimentation (lanes 2 and 5) and subjected to crosslinking with BMH (lanes 3 and 6). The crosslink to Asterix is indicated. d, FLAG or twin-Strep tag (TST) containing membrane-inserted RNCs of Rho^exttruncated 70 amino acids (aa) beyond TMD1 were subjected to fractionation and affinity purification. The total IVT products were centrifuged to obtain a membrane fraction, which was then solubilised under non-denaturing conditions (soluble fraction). The soluble fraction was then subjected to anti-FLAG affinity purification. Aliquots of the purification (with five-fold more loaded for the elution fraction) were analysed by anti-FLAG immunoblot (top panel). The bottom panel shows the elution fractions of the two purifications immunoblotted for CCDC47, Asterix, RPL8, Sec61β, and EMC2. Serial dilutions of ER microsomes were analysed in parallel. RNCs of Rho^extcan be efficiently affinity-purified, recovering the associated PAT complex. Note that the left and right lanes of the CCDC47 and Asterix blots are from the same gel and taken from the same exposure, with the vertical line indicating the point where the lanes were spliced together. e, Membrane-inserted RNCs of bovine preprolactin (pPL) truncated 56 residues beyond the signal sequence³⁴and Rho^exttruncated 70 residues beyond TMD1 were subjected to anti-FLAG affinity purification as in panel d. The TST-tagged Rho^ext served as a specificity control. Where indicated, the sample was crosslinked with BMH just prior to solubilisation of RMs. The top panel shows the anti-FLAG immunoblot of the steps of affinity purification (as in panel d), and the bottom panel shows the elution fractions of each purification immunoblotted for CCDC47, Asterix, RPL8, and Sec61α. The blots indicate that the pPL translation intermediate does not associate with the PAT complex, while the PAT complex is recovered with Rho^ext with comparable efficiency without or with crosslinking, which proved to be nearly quantitative as judged by the near absence of non-crosslinked Asterix. This indicates that association of the PAT complex with Rho^ext RNCs is salt- and detergent-resistant under the purification conditions employed. The faint band in the final lane of the CCDC47 is IgG heavy chain contamination. f, The top panel shows a diagram depicting the construct encoding full-length N-terminal FLAG-tagged TRAM2, whose topology is opposite to that of Rho. The bottom panel shows an experiment analogous to that for Rho^extshown in Fig. 1b. The purified samples were analysed by immunoblot adjacent to serial dilutions of RMs. Equal translation levels and recovery of RNCs is reflected by both the substrate blot and by similar levels of Sec61 subunits and the ribosome. A reaction lacking mRNA served as a negative control for non-specific binding to the affinity resin. Note that as for Rho^ext (Fig. 1b), recruitment of the PAT complex to TRAM2 translation intermediates is dependent on nascent chain length. Although a small amount of recruitment is detectable at lengths of 70-100 aa beyond TMD1, the initial point of stable maximal recruitment is observed at 110 aa beyond TMD1. g, Diagrams showing the substrate configurations, approximately to scale, for Rho and TRAM2 at the point of initial stable recruitment of the PAT complex. This corresponds to ~70 aa beyond TMD1 for Rho and ~110 aa beyond TMD1 for TRAM2. The difference in length can be accounted for by the different topology of TMD1. The translocon is not shown for simplicity.