EVIDENCE FOR A LOOP-LIKE INSERTION MECHANISM OF PRO-OMP-A INTO THE INNER MEMBRANE OF ESCHERICHIA-COLI

We have studied the insertion of pro-OmpA into the Escherichia coli me mbrane in vivo using various mutants that have either alterations in t he amino-terminal parts of the signal peptide or in the mature region that flanks the signal peptide. A pro-OmpA mutant with an amino termin al extension of 142 residues derived from ribulokinase (AraB) was anal ysed for its membrane insertion. The AraB portion, which includes a cl uster 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. Fur ther mutants of OmpA were constructed in the carboxy-terminal region f lanking the signal sequence. Pro-OmpA does not translocate across the membrane when a charge cluster, comprised of Lys-Arg-Arg-Glu-Arg, is i ntroduced after positions 5, 11 or 15 of the mature region, but is tra nslocated when the cluster is introduced after position 22. This defin es a region of about 20 residues in the mature part of pro-Ompa that i s crucial for membrane insertion. These results suggest that in the ca se of the Sec-dependent pro-OmpA, as with the Sec-independent M13 proc oat, the precursor assumes a loop-like structure involving the signal peptide and the early part of the mature region, leaving the amino ter minus of the signal peptide at the cytoplasmic face.