Fig. 2. Insertion competence and size of the cytosolic R4op complex. (A) Time dependence of R4op insertion into RMs. R4op was synthesised in the RRL, translation was stopped by the addition of puromycin and ribosomes were removed by sedimentation. Reactions were further incubated for the times indicated. RMs were then added and incubation continued for 30 minutes. R4op was immunoprecipitated and characterised by SDS-PAGE and autoradiography. The amounts of immunoprecipitated non-glycosylated R4op (black bars) and glycosylated g-R4op (grey bars) were quantified (see histogram). (B) Sucrose-density-gradient analysis of cytosolic R4op. R4op was synthesised in the RRL. Aliquots were loaded on top of 10-20% sucrose density gradients containing 2 mM ATP (left) or ADP (right). After centrifugation and fractionation, proteins were analyzed by SDS-PAGE and autoradiography. Black arrowheads and numbers above the gel indicate migration positions of proteins used as molecular markers and their molecular mass in kDa, respectively.