A genetic engineering approach to study the mode of assembly of the OmpF porin in the envelope of E coli.
Inducible hybrid genes encoding two large domains, a periplasmic domain consisting of the PhoS sequence and an outer membrane domain corresponding to various lengths of the OmpF mature sequence were constructed. The synthesized hybrid polypeptides are correctly processed during the early times of induction, their precursor forms being accumulated at later times. These hybrids restore sensitivity toward colicin A to ompF E coli B strain which suggests an outer membrane location. At least 2 of them are indeed localized in the outer membrane after immunogold labelling on ultrathin cryosections. Insertion of a hydrophobic sequence between PhoS and OmpF improves the trimerization and the assembly of the OmpF part. Only the hybrids presenting the last C-terminal 29 residues of OmpF are able to promote the colicin N killing action and to exhibit a trimeric conformation which is recognized by specific antibodies. Moreover, the deletion of the C-terminal region impairs the functional insertion of the OmpF domain; this indicates that the last membrane-spanning region of OmpF is necessary for the correct folding and orientation of the protein in the outer membrane.
Bolla
J M
JM
Centre de Biochimie et de Biologie Moléculaire, CNRS, Marseille, France.
Bernadac
A
A
Lazdunski
C
C
Pages
J M
JM
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003087:Colicins / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005818:Genetic Engineering; D007150:Immunohistochemistry; D018272:Porins; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014357:Trypsin
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D018272:Porins; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C028593:colicin receptor, E coli; D014357:Trypsin