Abstract
Like several other large, multimeric bacterial outer membrane proteins (OMPs), the assembly of the Klebsiella oxytoca OMP PulD does not rely on the universally conserved β-barrel assembly machinery (BAM) that catalyses outer membrane insertion. The only other factor known to interact with PulD prior to or during outer membrane targeting and assembly is the cognate chaperone PulS. Here, in vitro translation-transcription coupled PulD folding demonstrated that PulS does not act during the membrane insertion of PulD, and engineered in vivo site-specific cross-linking between PulD and PulS showed that PulS binding does not prevent membrane insertion. In vitro folding kinetics revealed that PulD is atypical compared to BAM-dependent OMPs by inserting more rapidly into membranes containing E. coli phospholipids than into membranes containing lecithin. PulD folding was fast in diC14:0-phosphatidylethanolamine liposomes but not diC14:0-phosphatidylglycerol liposomes, and in diC18:1-phosphatidylcholine liposomes but not in diC14:1-phosphatidylcholine liposomes. These results suggest that PulD efficiently exploits the membrane composition to complete final steps in insertion and explain how PulD can assemble independently of any protein-assembly machinery. Lipid-assisted assembly in this manner might apply to other large OMPs whose assembly is BAM-independent.
Highlights
IntroductionTheir assembly might rely on a different membrane insertion mechanism that, regardless of the transmembrane secondary structure, prevents the formation of a large open channel in the membrane
We previously showed that this truncated secretin, PulD28–42/259–660, folds via a multistep mechanism: membrane adsorbed monomers dodecamerise into a prepore that inserts into the membrane[29]
This report examines the factors required for the folding and assembly of the outer membrane proteins (OMPs) PulD, whose biogenesis is independent of the general OMP-specific assembly machinery (BAM)[23]
Summary
Their assembly might rely on a different membrane insertion mechanism that, regardless of the transmembrane secondary structure, prevents the formation of a large open channel in the membrane. We address this question using the secretin PulD as a model system. To address whether PulS has additional roles besides outer membrane targeting and how PulD overcomes the energetic barrier for efficient assembly, we took advantage of the spontaneous in vitro folding of PulD in a coupled transcription-translation reaction containing liposomes. We use the terms ‘folding’ and ‘assembly’ to distinguish between the in vitro and the in vivo processes, respectively
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