Escherichia coli sec mutants accumulate a processed immature form of maltose-binding protein (MBP), a late-phase intermediate in MBP export.
Protein translocation across the Escherichia coli cytoplasmic membrane may consist of several temporally or topographically distinct steps. Although early events in the translocation pathway have been characterized to some extent, the mechanisms responsible for the trans-bilayer movement of a polypeptide are only poorly understood. This article reports on our attempts to dissect the translocation pathway in vivo. A processed form of maltose-binding protein (MBP) was detected in the spheroplasts of secY and secA temperature-sensitive mutant cells that had been pulse-labeled at the permissive temperature (30 degrees C). This species of molecule was found to have an electrophoretic mobility identical to that of the mature MBP, but a considerable fraction of it was inaccessible to externally added protease. It had not attained the protease-resistant conformation characteristically observed for the exported mature protein. The radioactivity associated with this species decreased during chase and was presumably converted into the exported mature form, a process that required energy, probably the proton motive force, as demonstrated by its inhibition by an energy uncoupler. The spheroplast-associated processed form was more predominantly observed in the presence of a low concentration of chloramphenicol. A similar intermediate was also detected for beta-lactamase in wild-type cells. These results suggest that in a late phase of translocation, the bulk of the polypeptide chain can move through the membrane in the absence of the covalently attached leader peptide, and the secA-secY gene products are somehow involved in this process. We termed the processed intermediates processed immature forms.
Institute for Virus Research, Kyoto University, Japan.
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D018528:ATP-Binding Cassette Transporters; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D002462:Cell Membrane / Q000378:metabolism; D002701:Chloramphenicol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010446:Peptide Fragments / Q000302:isolation & purification; D014357:Trypsin
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; C461599:maltose transport system, E coli; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D002701:Chloramphenicol; D008320:Maltose; D014357:Trypsin