The Sec pathway facilitates integration of the TM123-AmiA fusion protein into inverted membrane vesicles. (A) The TM123-AmiA fusion co-sediments with IMVs containing elevated levels of SecYEG. Constructs TM123-AmiA and TM12_KK3-AmiA were synthesized in vitro in the presence of [³⁵S]methionine and either buffer alone (no), or IMVs (to a final concentration of 0.2 mg/ml protein) derived from strain SF100 harboring plasmid pET302 (WT) or pET2302 that overproduces SecYEG (YEG⁺⁺). The synthesis reaction was performed for 20 min at 37°C. 10% of the reaction mixture was removed to confirm that protein synthesis was similar between the different samples (lanes labeled “control”). The remaining reaction mixture was treated with 6 M urea (to remove peripherally bound protein), and the vesicles were sedimented by high-speed centrifugation. The pelleted vesicles were resuspended in Laemmli buffer (lanes labeled cosediment), and samples were analyzed by SDS-PAGE and autoradiography. (B) Proteinase K digestion of IMVs containing TM123-AmiA. Samples were prepared as described in A except that instead of sedimenting the samples they were treated with proteinase K. The arrow indicates the full-length protein and the arrowheads indicate protease-resistant fragments. (C) Predicted topology of TM123-AmiA in IMVs. The arrows indicate likely proteinase K–sensitive sites in the linker region between TMs 2 and 3.