Fig. 2: Substrate-directed assembly of the multipass translocon.

a, Templates used to generate truncated, Flag-tagged constructs for affinity purification of stalled (no stop codon) ribosome–nascent chain complexes. All stalled intermediates are appended with Met-Leu-Lys-Val. Luminal loops (grey) and native N-glycosylation acceptor sites (black circles) are indicated. b, Stalled, Flag-tagged ASGR1(N79A) and YIPF1 constructs truncated at the indicated positions were translated in rabbit reticulocyte lysate (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 ribosome–nascent chain complexes were isolated by sedimentation and analysed by SDS–PAGE and immunoblotting. Note the earliest intermediates (ASGR1 61-mer and YIPF1 140-mer) do not target to the membrane as their first TMD remains buried inside the ribosome exit tunnel, thus serving as a control for nonspecific binding. c, Stalled, Flag-tagged YIPF1 constructs truncated at positions 183 and 277 were translated in RRL in the presence of wild-type (WT), double-knockout (TMCO1/Nicalin (ΔTN) and TMCO1/CCDC47 (ΔTC)) or STT3A-knockout (ΔS) rough microsomes, and analysed as in b. d, Diagram of translocon composition at different stages of YIPF1 synthesis, based on data in b,c. e, Series of two-TMD YIPF1 templates containing different TMD1 and TMD2 sequences. The calculated apparent free energy of membrane insertion⁴⁸ (ΔG_app) for each TMD is indicated. f, Stalled, Flag-tagged YIPF1 constructs as in e were analysed as in b; quantification for n = 5 biological replicates is shown in Extended Data Fig. 4b.