Fig. 3.
Membrane targeting potential of TMS of type III-secreted transmembrane proteins (a) Calculation of ∆Gapp for the SRP-targeting window of 12–17 aa (∆GappSRP) for transmembrane proteins of type III-secreted transmembrane proteins (red) compared to E. coli transmembrane proteins. The classification of E. coli membrane proteins is according to Schibich et al.:17 SRP substrates (dark gray), non SRP substrates (middle gray) and substrates, in which the first TMS was skipped by SRP (light gray). b, c Relevant TMS of T3SS substrates were assessed for their inner membrane targeting potential and membrane integration in a S-35 Met-based pulse-chase targeting assay using inverted leader-peptidase (Lep-inv, b) and ProW Nt/TM1/P2 (c), respectively23. The principle of the assays is shown on the left. Membrane-inserted Lep-inv/ProW Nt/TM1/P2 were cleaved into a smaller fragment by exogenously added proteinase K. Lep-inv/ProW Nt/TM1/P2 that fails to insert into the membrane is not affected by proteinase K. Cleavage can be detected by immunoprecipitation. For assessment of targeting, the H1 segment of Lep-inv or ProW Nt/TM1/P2, respectively, was exchanged against the indicated segment of interest. Proteins were expressed in E. coli MC4100 from rhamnose-inducible plasmids. After spheroplasting and addition of proteinase K, proteins of interest were immunoprecipitated and analyzed by SDS PAGE and autoradiography of 35-S. The outer membrane protein OmpA served as control for successful proteinase K digestion. Band X (indicated by asterisk) was used as a control for intact spheroplasts. A representative result of three independent experiments is shown. IM inner membrane, TMS transmembrane segment