FIGURE 3.

S1R biotinylates components of the ER translocation complex. (A) A schematic of the components involved in translocation of proteins into the ER. (B) A list of ER translocation components identified in the S1R proximatome. (C) Biotinylated proteins were purified from HEK293T cells stably expressing either GFP-Apex or S1R-Apex that were untreated or treated with either Haloperidol or (+)-PTZ. The proteins were purified using streptavidin conjugated beads. The bound proteins were eluted and analyzed by Western blotting using an antibody against Sec61alpha. S1R-Apex expressed in HEK293T cells biotinylates Sec61alpha. (D) HEK293T cells were transiently transfected with a construct expressing either GFP-Apex or Sec61 beta-Apex. Biotinylated proteins were purified using streptavidin conjugated beads, the bound proteins were eluted and analyzed by Western blotting using an antibody against endogenous S1R. Endogenous S1R is biotinylated by Sec61 beta-Apex, a component of the ER translocation complex. (E) HEK293T cells stably expressing GFP or S1R-Apex were either untreated (DMSO) or treated with the drug Eeyarestatin. Eeyarestatin blocks the transport of proteins across the Sec61 translocation channel. Biotinylated proteins were purified using streptavidin conjugated beads and the bound proteins were eluted and analyzed by Western blotting using antibodies against Sec61 alpha and Calnexin. (F) The above experiment was repeated in triplicate and the binding of S1R-Apex with Sec61 alpha and Calnexin was quantified. Unpaired t tests were used for these analyses; ****p < 0.0001, ns = not significant. Blocking protein transport across the Sec61 channel disrupts the proximal association between S1R and Sec61 alpha.