Alterations to the signal peptide of an outer membrane protein (OmpA) of Escherichia coli K-12 can promote either the cotranslational or the posttranslational mode of processing.
The signal sequence of the precursor of the Escherichia coli outer membrane protein OmpA was altered by oligonucleotide insertions into the corresponding gene. In one case, OmpA-S1, the hydrophobic core of the signal peptide, is reduced from 12 to 10 residues, and one positive charge is added near the NH2-terminus. In another case, OmpA-P1, the hydrophobic core is extended from 12 to 16 residues. The pro-OmpA protein is normally processed partially co- and partially posttranslationally. Processing of the pro-OmpA-S1 protein was entirely posttranslational and that of the pro-OmpA-P1 protein strictly cotranslational. Evidence is presented which strongly suggests that posttranslational processing reflects posttranslational translocation across the plasma membrane. The generation times of cells expressing pro-OmpA-P1 or pro-OmpA-S1 were identical and the pro-OmpA-S1 polypeptide could be chased into the mature protein in the absence of protein synthesis. Hence, it does not matter which mode of processing, or rather translocation, is used. The same oligonucleotides were inserted into the ompA gene of plasmid pRD87; a plasmid which leads to overproduction of the protein and to massive accumulation of both the mature protein and the precursor. In the OmpA signal sequence encoded by pRD87-P2 the hydrophobic core is extended from 12 to 20 residues. This peptide was also rapidly removed. Therefore, regardless of whether the hydrophobic core contains 12, 16, or 20 lipophilic residues, not only does the signal sequence always function correctly to mediate export, but in each case, the cleavage site is always accessible to the signal peptidase.
Freudl
R
R
Max-Planck-Institut für Biologie, Tübingen, Federal Republic of Germany.
MacIntyre
S
S
Degen
M
M
Henning
U
U
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D021382:Protein Sorting Signals