FIG 4 .

Analysis of in vitro-synthesized PilQ, XcpQ, and PscC insertion into liposomes. Secretins PilQ (A), XcpQ (B), and PscC (C), lacking signal sequences, were synthesized in vitro in the presence of asolectin liposomes. Samples were analyzed by SDS-PAGE and immunoblotting with the appropriate secretin-specific antisera and with or without prior urea treatment to dissociate peripherally associated proteins and phenol treatment to dissociate multimers. Equivalent amounts of each sample in relation to the amount of total reaction mixture represented in lane 1 were loaded. DNA encoding green fluorescent protein (Gfp) was used in a control reaction for proteins cross-reacting with the antisera. Lane 1, total in vitro reaction; 2, centrifuged liposomes; 3, liposomes heated to 100°C in SDS; 4, phenol-treated liposomes; 5, urea-washed liposomes; 6, urea-washed liposomes treated with phenol; 7, total in vitro reaction of Gfp control; 8, centrifuged liposomes of Gfp control. Transmission electron microscope images of negatively stained liposomes, after being washed with 4 M urea, are shown on the right in each panel. The white arrows indicate top views, and the black arrow indicates a side view. Bar, 100 nm.