Stepwise movement of preproteins in the process of translocation across the cytoplasmic membrane of Escherichia coli.
Derivatives of proOmpA possessing the second cysteine residue at position +302 and the first one at different positions were constructed at the DNA level. They were oxidized to form disulfide-bridged loops of different sizes at different positions. In the presence of a protonmotive force, proOmpAs possessing a smaller loop could be translocated across the membrane in vitro, whereas ones possessing loops comprising more than 16 amino acid residues were hard to translocate. The sizes of polypeptide chains that had been translocated and had become protease-resistant were determined in both the presence and absence of the protonmotive force. The size was the same for all proOmpAs possessing the first cysteine residue between +244 (proOmpA L59) and +274 (proOmpA L29). When the first cysteine residue was moved further away from the N terminus, a sudden increase in size, of approximately 30 amino acid residues, was observed, the size being the same for proOmpAs possessing the first cysteine residue between +278 (proOmpA L25) and +293 (proOmpA L10). The shift in size between proOmpA L29 and proOmpA L25 was observed with different proteases exhibiting different substrate specificities. Treatment with these proteases resulted in complete digestion of SecA on everted membrane vesicles, whereas Sec proteins integrated into membranes were considerably resistant to the treatment. These results can be best interpreted as that the translocation of preproteins through the secretory machinery takes place in every 30 amino acid residues and that SecA is responsible for the stepwise movement.
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
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Uchida
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Mori
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Mizushima
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; C057184:outer membrane protein A precursor (E coli)