YidC also facilitates integration of the TM123-AmiA fusion protein into inverted membrane vesicles. (A) The TM123-AmiA fusion cosediments with IMVs containing elevated levels of YidC. 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 0.4 mg/ml protein) derived from strain FTL10 grown under conditions where the chromosomal copy of yidC was depleted (YidC⁻) or the same conditions but where YidC was co-overproduced from a plasmid (YidC⁺). 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 the 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 reaction was stopped by addition of Laemmli buffer and samples were analyzed by SDS-PAGE and autoradiography. The arrows indicate the full-length protein and the arrowheads indicate protease-resistant fragments.