Figure 8. Topological analysis and membrane integration of Sco3746TMD-MBP and PFDTMD-MBP.
(A) Membrane (M; 100 µg protein) and soluble (S; 50 µg protein) fractions of E. coli HS3018-A (△malE, tat+) and HS3018-A△tat strains harboring pSU18 (empty vector), pSU18 encoding Sco3746TMD-MBP or PFDTMD-MBP fusion proteins, or variants of these where the twin-arginine motif was substituted to twin-lysine were separated by SDS-PAGE (12% acrylamide), transferred to nitrocellulose membrane and immunoblotted with an anti-MBP antibody. (B) Crude membranes of the same strains and plasmids were treated with 4M urea or 0.2M carbonate, and the presence of the fusion proteins in the wash supernatant (S) and pelleted membrane (M) was analyzed by immunoblotting as in (A). (C) Predicted locations of cysteine substitutions of Sco3746TMD-MBP used for topology analysis. (D) Cell suspensions of strain HS3018-A harboring pSU18 alone (empty vector), or pSU18 encoding Sco3746TMD-MBP or the indicated single cysteine substitutions of Sco3746TMD-MBP were incubated with buffer alone, with 5 mM MAL-PEG, or were lysed by sonication and incubated with 5 mM MAL-PEG. Subsequently all samples were quenched, lysed and membranes pelleted by ultracentrifugation. Membrane samples (150 µg of protein) were separated by SDS PAGE and immunblotted as in (A).