title: Overproduction of the proFhuA outer membrane receptor protein of Escherichia coli K-12: isolation, properties, and immunocytochemical localization at the inner side of the cytoplasmic membrane.
abstract: The fhu operon of Escherichia coli K-12 comprises four genes, termed fhuA,C,D,B, which are involved in the uptake of iron-hydroxamate compounds. The fhuA gene encodes the outer membrane receptor protein. Cells that contained three copies of the fhuACD fragment on the thermoamplifiable plasmid pHK232 accumulated at 37 degrees C large amounts of the proFhuA protein. Most of the overproduced proFhuA protein was not translocated into the outer membrane but instead precipitated at the cytoplasmic side of the inner membrane, presumably at the sites of synthesis. Despite inhibition of export proFhuA synthesis continued. The precipitate formed was sedimented by centrifugation at 8,000 x g. The proFhuA protein could be solubilized in 1% sodium dodecyl sulfate. Replacement of sodium dodecyl sulfate by Triton X-100 resulted in a proFhuA protein which exhibited 10% of the phage T5 binding activity of renatured mature FhuA protein. Binding of the phage T5 was inhibited by the FhuA-specific ligands ferrichrome, albomycin and colicin M. Limited proteolysis of the isolated pro- and mature form of the FhuA protein with trypsin yielded similar oligopeptide patterns. Addition of ferrichrome affected trypsin cleavage of both proteins in the same way. The common proteolytic intermediates together with phage inactivation indicate a similar conformation of the pro- and mature form.
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002462:Cell Membrane / Q000032:analysis; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007158:Immunologic Techniques; D008854:Microscopy, Electron; D010957:Plasmids; D011991:Receptors, Virus / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D011991:Receptors, Virus; D014357:Trypsin