Fig. 3: SARS-CoV-2 proteins are variably dependent on the Sec61 complex and/or the EMC for ER membrane translocation/insertion.

(A) Schematic of the in vitro ER import assay in which control SP cells, or those depleted of a subunit of the Sec61 complex and/or the EMC via siRNA, are used as a source of ER membrane. Following translation, OPG2-tagged translation products (i.e. membrane-associated and non-targeted nascent chains) are recovered by immunoprecipitation, resolved by SDS-PAGE and analysed by phosphorimaging. OPG2-tagged variants of the SARS-CoV-2 (B) spike (S-OPG2), (C) ORF8 (ORF8-OPG2), (D) envelope (OPG2-E) and (E) ORF6 (OPG2-ORF6-OPG2, different Gly species labelled as described in the legend to Fig. 2) proteins were synthesised in rabbit reticulocyte lysate supplemented with control SP cells (lanes 1–2) or those with impaired Sec61 complex and/or EMC function (lanes 3–6). Radiolabelled products were recovered and analysed as described in (A). Membrane translocation/insertion efficiency was determined using the ratio of the N-glycosylation of lumenal domains, identified by treatment with Endo H (EH, lane 1), relative to the NT control (set to 100% translocation/insertion efficiency). Quantifications are given as means ±SEM for independent translation reactions from separate siRNA treatments performed in triplicate (n=3) and statistical significance (two-way ANOVA, DF and F values shown in the figure) was determined using Dunnett’s multiple comparisons test. Statistical significance is given as: n.s., non-significant >0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.