Fig. 5.

Interaction of the HTL with ribosomes supports membrane protein insertion. (A) Binding of detergent-solubilized HTL and SecYEG to 70S ribosomes followed by fluorescence intensity. Constant amounts of SecYEG and HTL containing SecY labeled with Cy3 at positions 148 and 215 were exposed to increasing concentrations of 70S ribosomes, leading to an increased fluorescence in the case of SecY labeled at position 148, but not at 215. (B) In vitro synthesized cyoA mRNA was incubated together with empty liposomes or proteoliposomes containing YEG or the HTL together with an E. coli S30 membrane-free cell extract including single-chain signal recognition particle-SRP receptor (scSRP) (39) to assess incorporation of ³⁵S-labeled CyoA directly into an in vitro membrane bilayer. Values represent the mean of four independent experiments ± SEM. (C) Representative phosphorimaging of trypsin/urea-treated ³⁵S-labeled CyoA. Arrowhead represents full-length CyoA; asterisk denotes protease-sensitive fragment.