Modulation of the effects of mutations in the basic region of the OmpA signal peptide by the mature portion of the protein.
Oligonucleotide-directed site-specific mutagenesis was used to study the structure-function relationship of the positively charged amino terminus of the Escherichia coli outer membrane protein OmpA signal peptide. Mutations were isolated which reduced the overall charge of the amino-terminal region from +2 (wild type) to +1, 0, and -1, as well as one mutation from Thr to Ser at position 4. DNA encoding the wild type and mutant OmpA signal peptides was then fused in-frame to DNA encoding the mature regions of Staphylococcus aureus nuclease A and TEM beta-lactamase. In the case of both the beta-lactamase and nuclease fusions, normal processing was no longer observed when the charge at the amino terminus was reduced to zero or made negative. Differences between the two hybrid proteins were observed in the case of the Thr to Ser mutation. As expected, this mutation had no effect on the beta-lactamase hybrid; however, the processing rate of the nuclease hybrid protein was reduced to nearly one-half. Furthermore, this effect was essentially reversed when a Lys residue at position 3 was deleted. A model is presented which explains the differing effects of a signal peptide mutation on the secretion of different hybrid proteins based on kinetic differences in the translocation of the nuclease and beta-lactamase proteins.
Lehnhardt
S
S
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry, Piscataway, New Jersey 08854-5635.
Pollitt
N S
NS
Goldstein
J
J
Inouye
M
M
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals / Q000378:metabolism*; D001618:beta-Lactamases / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D021382:Protein Sorting Signals; D001618:beta-Lactamases