Export of a protein into the outer membrane of Escherichia coli K12. Stable incorporation of the OmpA protein requires less than 193 amino-terminal amino-acid residues.
The cloned ompA gene encoding the major outer membrane protein OmpA of Escherichia coli has been shortened in vitro by exonuclease digestion from the end corresponding to the CO2H terminus of the protein. Nine derivatives were identified which still possessed substantial parts of the ompA gene and one was constructed which had suffered a small deletion early in the gene. Gene fragments encoding NH2-terminal OmpA sequences of 45, 133, 193, and 227 residues of the 325 amino acids of OmpA were examined in detail at the DNA level and for OmpA protein fragments synthesized. The latter two fragments were incorporated into the outer membrane and all known functions of the OmpA protein were expressed whereas the fragment with 133 OmpA-specific residues was not stably incorporated into this membrane. In all cases where OmpA functions were observed, an OmpA-specific polypeptide of Mr 24 000 was found in cell envelopes, regardless of the size of the residual ompA sequences and of the fused coding sequences in the vector DNA. Pulse-label experiments revealed larger initial translation products, most of which were degraded to the protein of Mr 24000. The 133-residue OmpA fragment was also detected but proved to be entirely unstable. It is argued that the OmpA protein consists of two domains and that the NH2-terminal moiety from residues 1 to about 180 represents the membrane domain of the polypeptide. Therefore, the loss of about 50, possibly less, CO2H-terminal residues from this domain suffices to interfere with stable incorporation into the outer membrane.
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000378:metabolism*; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D010957:Plasmids
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008565:Membrane Proteins