Extended Data Figure 8: Insertase-disrupting mutation prevents Get1/2 TMD interactions with pre-integrated tail-anchored proteins.

a, Affinity-purified Get3–Sec22_S192Cs was road-blocked with S protein and incubated with the indicated microsomes for subsequent crosslinking analysis as in Fig. 3c. b, NEM alkylation or mock treatment of the indicated microsomes was followed by NEM quenching and membrane solubilization in SDS. The denatured samples were subjected to PEG-maleimide alkylation, as indicated. Alkylation was quenched and samples were subjected to SDS–PAGE analysis and visualized by immunoblotting. Get2-1scPEG indicates the PEGylated form of Get2-1sc. Quantitation revealed that the cysteine accessibility factor of T15C was 0.97 for Get2-1sc and 0.52 for Get2-1_TM3msc. Thus, the lack of Sec22_S192C crosslinking to Get2-1_TM3sc (T15C) (Extended Data Fig. 8a) is unlikely owing to cysteine inaccessibility. c, Affinity-purified Get3–Sec22_S192Cs was road-blocked with S protein for 10 min at room temperature or mock-treated before incubation with Get1_T15C microsomes for the indicated amounts of time. Samples were simultaneously subjected to Sec22 insertion and crosslinking analysis as in Figs 1b and 3c, respectively. On the right is a kinetic analysis plot of the gel data shown on the left. XL indicates the amount of crosslinked product between Get1 and Sec22. d, Affinity-purified Get3–Sbh1_L58Cs was incubated with the indicated microsomes at room temperature for 5 min. Samples were simultaneously subjected to substrate insertion and crosslinking analysis as in Figs 1b and 3c, respectively. e, Cell extracts containing Get3–Sbh1_L158Cs were incubated with the indicated microsomes for 5 min at room temperature and crosslinking/immunoprecipitation analysis as in Extended Data Fig. 7b.