Insertion of a signal peptide-derived hydrophobic segment into the mature domain of OmpC, an outer membrane protein, does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E. coli.
The effects of a hydrophobic peptide segment inserted into the amino-terminal region of the mature domain of OmpC, an outer membrane protein, on its translocation across the cytoplasmic membrane was studied. Both the intact OmpC and central domain-deleted OmpC were examined. The hydrophobic segment was derived from the signal peptide of OmpF. Secretory translocation across the cytoplasmic membrane was examined by means of proteinase K treatment. Four monoclonal antibodies that recognize different regions of OmpC were used to characterize proteinase K-resistant fragments. Insertion of the hydrophobic segment did not appreciably prevent the translocation of these proteins across the cytoplasmic membrane, larger parts of them being found as mature forms, which were mostly localized outside the cytoplasmic membrane. Circumstantial evidence supports the view, on the other hand, that the inserted hydrophobic domain was retained in the cytoplasmic membrane. It is concluded, therefore, that the hydrophobic segment, although it is not exported across the cytoplasmic membrane, does not prevent the secretion of the following polypeptide chain. The secretion was dependent on the amino-terminal signal peptide. Insertion of positive charges immediately after the hydrophobic segment resulted in suppression of the translocation. Based on these results possible mechanisms by which the secretion of the polypeptide chain after the hydrophobic segment are discussed.
Shinkai
A
A
Yamada
H
H
Mizuno
T
T
Mizushima
S
S
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003593:Cytoplasm / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D010455:Peptides; D021382:Protein Sorting Signals