Mechanism of localization of major outer membrane lipoprotein in Escherichia coli. Studies with the OmpF-lipoprotein hybrid protein.
A chimera gene consisting of the ompF promoter, the coding regions for the signal peptide and the NH2-terminal 11 amino acid residues of outer membrane OmpF protein, and the coding region for the major outer membrane lipoprotein devoid of the NH2-terminal 7 amino acid residues was constructed. Escherichia coli carrying the cloned chimera gene produced a hybrid protein with the predicted chemical structure. The protein was localized in the periplasmic space with an interaction with the peptidoglycan layer. These results indicate that the hybrid protein was expressed, secreted across the cytoplasmic membrane, and processed for the signal peptide normally. The hybrid protein, however, was not incorporated into the outer membrane, suggesting the importance of the lipid domain in the assembly of the lipoprotein into the outer membrane. Although a larger part of the protein was extractable with sodium dodecyl sulfate, a part of the hybrid protein was covalently bound to the peptidoglycan layer as the lipoprotein is. Upon treatment with lysozyme of the envelope the hybrid protein became water soluble. The solubilized protein most probably existed as a trimer. These results most likely suggest that the major lipoprotein exists as a trimer in the periplasmic space with interactions with the peptidoglycan layer through the protein domain on one side and with the outer membrane through the lipid domain on the other side.
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins; D001483:Base Sequence; D002678:Chimera; D004262:DNA Restriction Enzymes; D004120:Dimethyl Suberimidate; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000235:genetics*; D008970:Molecular Weight; D009876:Operon; D010957:Plasmids; D055503:Protein Multimerization
D001425:Bacterial Outer Membrane Proteins; D008565:Membrane Proteins; D004120:Dimethyl Suberimidate; D004262:DNA Restriction Enzymes