A proline residue near the amino terminus of the mature domain of secretory proteins lowers the level of the proton motive force required for translocation.
A large variety of proOmpF-Lpps, hybrid secretory proteins composed of the signal region of proOmpF and the mature part of the major lipoprotein, either possessing or not possessing a proline residue near the amino terminus of their mature domains, were constructed at a DNA level, and the rates of their in vitro translocation were determined in the presence and absence of the proton motive force (delta muH+). A proline residue at the signal peptide cleavage site (position +1) blocked the cleavage reaction but not the translocation reaction. All the proOmpF-Lpps examined exhibited approximately the same translocation rate in the presence of delta muH+ irrespective of the presence or absence of a proline residue near the amino terminus. In the absence of the delta muH+, which was achieved by either depletion of the respiratory substrate or the use of urea-treated membrane vesicles permeable to protons, proOmpF-Lpps possessing a proline residue near the amino terminus of the mature domain were translocated whereas those possessing no proline residue in this region were not translocated at all or only very weakly. The position of the proline residue was then moved stepwise away from the amino terminus of the mature domain. The further the position was moved away, the slower was the rate of translocation in the absence of delta muH+. The removal of the proline residue at position +2 of the mature domain of proOmpA also made the delta mu(H+)-independent translocation appreciably slower. It is suggested that the conformational flexibility endowed by the proline residue on the junction region between the signal peptide and the mature domain allows the translocation in the absence of delta muH+ and that this junction region must take on a particular conformation for initiation of the translocation reaction.
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Institute of Applied Microbiology, University of Tokyo, Japan.
Yamada
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Mizushima
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D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D004247:DNA / Q000737:chemistry*; D008074:Lipoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011392:Proline / Q000378:metabolism*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism; D011522:Protons; D011994:Recombinant Proteins; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D008074:Lipoproteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011522:Protons; D011994:Recombinant Proteins; C068862:proOmpF-Lpp secretory protein, recombinant; D004247:DNA; D011392:Proline