Figure 7. Mutations in the lumenal plug of Sec61α confer resistance to cotransins.
(A) Parental HCT-116 cells and resistant clones were treated with increasing concentrations of CT8 for 72 hr, and viability was assessed by the Alamar Blue assay (mean ± S.D., n = 4). (B) HEK293-FRT cells stably expressing wild-type or mutant Sec61α were transfected with a plasmid encoding TNFα and treated with increasing concentrations of CT8. After 24 hr, TNFα expression was analyzed by immunoblotting. (C) CT7 photo-crosslinking to recombinant wild-type and mutant FLAG-Sec61α. Microsomes were incubated with 250 nM CT7 in the presence or absence of excess CT9 (10 µM), photolyzed, and analyzed by in-gel fluorescence and immunoblotting as in Figure 1C. (D) Homology model of human Sec61α showing the location of cotransin resistance mutations (red) and the TMD docking site (yellow). Lateral gate helices colored as in Figure 4E.