In vivo effect of asparagine in the hydrophobic region of the signal sequence.
On the basis of the biophysical studies on the synthetic mutant (Ile-8----Asn) OmpA signal peptide in the preceding paper (Hoyt, D. C., and Gierasch, L.M. (1991) J. Biol. Chem. 266, 14406-14412), the in vivo effects of the same mutation were examined by fusing the mutant OmpA signal sequence to Staphylococcus aureus nuclease or TEM beta-lactamase. The mutation in which the isoleucine residue at position 8 of the OmpA signal sequence of Escherichia coli was replaced with a neutral polar residue, asparagine, resulted in a defective signal peptide. The mutant signal sequence was unable to be processed, and the precursor molecule accumulated in the cytoplasmic as well as in the membrane fractions, indicating that the Ile-8----Asn OmpA signal sequence is not competent for translocating nuclease A or beta-lactamase across the membrane. This result is consistent with the in vitro studies on the Ile-8----Asn OmpA signal peptide, which indicated that the mutant signal peptide was unable to penetrate into the hydrophobic core of the lipid bilayer. Other asparagine or glutamine substitution mutations in the hydrophobic region of the OmpA signal sequence were also examined. Interestingly, the OmpA signal sequence with either Ile-8----Gln, Val-10----Asn, or Leu-12----Asn mutation was completely defective as the Ile-8----Asn OmpA signal sequence, while the Ile-6----Asn and Ala-9----Asn OmpA nucleases were able to be processed to secrete nuclease, although the processing occurred at a much slower rate than the wild-type OmpA nuclease. These results indicate that the defects depend on the position of the lesion in the hydrophobic core of the OmpA signal sequence.
Goldstein
J
J
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
Lehnhardt
S
S
Inouye
M
M
D000595:Amino Acid Sequence; D001215:Asparaginase / Q000502:physiology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D021382:Protein Sorting Signals / Q000235:genetics*; D013211:Staphylococcus aureus / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D008051:Lipid Bilayers; D021382:Protein Sorting Signals; D001215:Asparaginase; D001618:beta-Lactamases