Fig. 3: E protein facilitates the membrane translocation process of TMED10-mediated UcPS.

a Diagram of in vitro translocation assay. In brief, proteoliposomes with GST-tagged Myc-E, TMED10 alone or both were incubated with recombinant mIL1β-FLAG protein at 30 °C for 1 h. Proteinase K digestion was performed to determine the amount of membrane-incorporated cargo. b In vitro membrane translocation of mIL1β-FLAG with control or GST-TMED10 proteoliposomes in the absence or presence of GST-Myc-E of SARS2. c Diagram of cell-free membrane translocation assay. In brief, membrane fraction collected from HEK293T cells expressing control (Ctrl), Myc-E-SARS2, TMED10-V5 or both after differential centrifugation was incubated with cytosol from HEK293T cells, recombinant mIL1β-FLAG plus GTP and ATPR for 1.5 h at 30 °C. After then, OptiPrep gradient ultracentrifugation was performed to remove the free proteins. Membrane floating on the top was collected and the translocated mIL1β was determined by proteinase K protection assay. d Cell-free membrane translocation of mIL1β-FLAG in the absence or presence of TMED10, Myc-E-SARS2 or both. The membrane fraction was collected from TMED10KO HEK293T cells. e Cell-free membrane translocation of mIL1β-FLAG in the absence or presence of Myc-tagged E of indicated coronaviruses. f, g Diagram of the GFP complementation assay (f). Cells expressing GFP (1–10)-TMED10 were transfected with negative control (NC) or mIL1β-GFP11 along without (Ctrl) or with Myc-tagged E of indicated coronaviruses. Complemented GFP signals were analyzed by FACS analysis and quantified in (g). Prot K, Protease K; TX-100, TritonX-100; ATPR, ATP regeneration system. Data are mean ± s.d. Statistical significance was assessed using one-way ANOVA (n = 5) followed by Tukey’s multiple-comparison test. P values are indicated. Source data are provided as a Source Data file.