Fig. 3.

TRC40 binds short secretory proteins. (A) Translocation-competent fractions of ppcecAOPG2 translations were pooled, and supplemented with ER-derived rough microsomes (RMs) where indicated. Selected samples were then treated with the bifunctional crosslinking reagent BMH, and adducts with TRC40 recovered by immunoprecipitation (cf. supplementary material Fig. S3). The resulting adducts were quantified by phophorimaging, and the value obtained in the presence of RMs was expressed as a percentage of that recovered in their absence. (B) In vitro translations of various precursors were performed as previously described in the presence or absence of recombinant TRC40. After synthesis was complete, the reactions were diluted with buffer and incubated with nickel agarose. The beads were recovered by centrifugation and washed repeatedly before any bound proteins were eluted. Radiolabelled products were analysed by SDS-PAGE and phosphorimaging. Brackets indicate unmodified radiolabelled protein eluted from TRC40.