title: Lipids assist the membrane insertion of a BAM-independent outer membrane protein.
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.
authors.affiliation: Institut Pasteur, Molecular Genetics Unit, Departments of Microbiology and of Structural Biology and Chemistry, rue du Dr. Roux, 75724 Paris Cedex 15, France, CNRS ERL3526, rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Gerard H M
authors.initials: GH
authors.lastname: Huysmans
authors.affiliation: Institut Pasteur, Molecular Genetics Unit, Departments of Microbiology and of Structural Biology and Chemistry, rue du Dr. Roux, 75724 Paris Cedex 15, France, CNRS ERL3526, rue du Dr. Roux, 75724 Paris Cedex 15, France.|Institut Pasteur, Laboratory of Macromolecular Structure and Signalling, Departments of Microbiology and of Structural Biology and Chemistry, rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Guilvout
authors.affiliation: C-CINA Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, CH 4058 Basel, Switzerland.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chami
authors.affiliation: Institut Pasteur, Molecular Genetics Unit, Departments of Microbiology and of Structural Biology and Chemistry, rue du Dr. Roux, 75724 Paris Cedex 15, France, CNRS ERL3526, rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Nicholas N
authors.initials: NN
authors.lastname: Nickerson
authors.affiliation: Institut Pasteur, Molecular Genetics Unit, Departments of Microbiology and of Structural Biology and Chemistry, rue du Dr. Roux, 75724 Paris Cedex 15, France, CNRS ERL3526, rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Pugsley
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*
chemical_list: D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers