Fig. 1. Post-translational membrane insertion of R4op. (A) Topology of RAMP4 in the ER membrane. RAMP4 is a tail-anchored ER membrane protein that exposes its N-terminus on the cytosolic and the C-terminus on the lumenal side of the membrane. (B) Schematic representation of RAMP4 and RAMP4op (R4op). R4op contains at its C-terminus a bovine opsin tag comprising 13 amino acid residues (dark grey box). The tag provides an N-glycosylation site (fork). The predicted transmembrane domain (TMD) is represented as a black box. A single cysteine residue in the TMD is typed in bold. (C and D) In vitro translation and membrane insertion of R4op and the type II membrane protein Invariant chain (Ii), respectively. Proteins were synthesised in rabbit reticulocyte lysate (RRL), in the absence (lanes 1, 4, 5 and 6) or presence (lanes 2 and 3) of rough microsomes (RM co). Rough microsomes were added after completion of translation (RM post) to samples shown in lanes 4 and 5. Where indicated, samples were treated with EndoH to remove N-linked oligosaccharides. Proteins were immunoprecipitated using either anti-opsin (C) or anti-Ii (D) antibodies, were separated by SDS-PAGE and visualised by autoradiography. g-R4op: glycosylated R4op; Ii^*: non-glycosylated Ii.