Abstract
RcsF (regulator of capsule synthesis) is an outer membrane (OM) lipoprotein that functions to sense defects such as changes in LPS. However, LPS is found in the outer leaflet, and RcsF was thought to be tethered to the inner leaflet by its lipidated N terminus, raising the question of how it monitors LPS. We show that RcsF has a transmembrane topology with the lipidated N terminus on the cell surface and the C-terminal signaling domain in the periplasm. Strikingly, the short, unstructured, charged transmembrane domain is threaded through the lumen of β-barrel OM proteins where it is protected from the hydrophobic membrane interior. We present evidence that these unusual complexes, which contain one protein inside another, are formed by the Bam complex that assembles all β-barrel proteins in the OM. The ability of the Bam complex to expose lipoproteins at the cell surface underscores the mechanistic versatility of the β-barrel assembly machine.
Highlights
RcsF is an outer membrane (OM) lipoprotein that functions to sense defects such as changes in LPS
OM lipoproteins are targeted to the Lol machine, which consists of an inner membrane ATP-driven LolCDE complex that extracts lipoproteins from the IM [7]; a periplasmic chaperone LolA, which escorts lipoproteins across the aqueous periplasmic space [8]; and an OM acceptor lipoprotein LolB, which anchors lipoproteins to the inner leaflet of the OM [9]
For that we generated two mutant RcsF proteins: one in which we introduced a Lol avoidance mutation (SM→DQ substitution at positions +2 and +3) that resulted in retention of RcsF in the inner membrane (RcsFIM) [17] and one in which we replaced the lipoprotein signal sequence of RcsF with the well-characterized signal sequence of OmpA, which resulted in production of RcsF lacking an N-terminal lipid (RcsFSS-OmpA)
Summary
We generated 30 functional RcsF-Strep variants with pBPA sites distributed along the linker as well as throughout the surface of the core domain and tested their ability to cross-link to OMPs by Western blot analysis (Fig. 2 and SI Appendix, Fig. S2). To identify residues of OMPs that interact with RcsF, we used several RcsF-Strep variants with pBPA at the linker sites followed by in vivo UV cross-linking, Strep-Tactin purification, SDS/PAGE with in-gel digestion, and high-resolution nano-flow UPLC-MS to sequence and map cross-linked peptides. This decrease was specific to RcsF/OmpA because RcsF/Lpp cross-linking did not decrease in these mutants Both bamA101 and bamA616 bamB are Rcs-activating mutations judged by rprA-lacZ reporter fusions, and they confer a mucoid phenotype. When OmpA was refolded in the PNAS | Published online September 29, 2014 | E4353
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call