Evidence for a Loop‐Like Insertion Mechanism of Pro‐Omp A into the Inner Membrane of Escherichia Coli

Abstract

We have studied the insertion of pro-OmpA into the Escherichia coli membrane in vivo using various mutants that have either alterations in the amino-terminal parts of the signal peptide or in the mature region that flanks the signal peptide. A pro-OmpA mutant with an amino terminal extension of 142 residues derived from ribulokinase (AraB) was analysed for its membrane insertion. The AraB portion, which includes a cluster of seven charged residues close to the signal sequence, did not interfere with the Sec components and allowed efficient export of OmpA. During translocation the AraB portion remained in the cytoplasm. Further mutants of OmpA were constructed in the carboxy-terminal region flanking the signal sequence. Pro-OmpA does not translocate across the membrane when a charge cluster, comprised of Lys-Arg-Arg-Glu-Arg, is introduced after positions 5, 11 or 15 of the mature region, but is translocated when the cluster is introduced after position 22. This defines a region of about 20 residues in the mature part of pro-OmpA that is crucial for membrane insertion. These results suggest that in the case of the Sec-dependent pro-OmpA, as with the Sec-independent M13 procoat, the precursor assumes a loop-like structure involving the signal peptide and the early part of the mature region, leaving the amino terminus of the signal peptide at the cytoplasmic face.