In vivo studies of the role of SecA during protein export in Escherichia coli.
SecA is found in Escherichia coli both tightly associated with the cytoplasmic membrane where it functions as a translocation ATPase during protein export and free in the cytosol (R. J. Cabelli, K. M. Dolan, L. Qian, and D. B. Oliver, J. Biol. Chem. 266:24420-24427, 1991; D. B. Oliver and J. Beckwith, Cell 30:311-319, 1982; W. Wickner, A. J. M. Driessen, and F.-U. Hartl, Annu. Rev. Biochem. 60:101-124, 1991). Here we show that SecA can be immunoprecipitated from the cytosol in complex with both fully elongated and nascent species of the precursor of maltose-binding protein, an exported, periplasmic protein. In addition, under conditions in which the distribution of SecA between the cytosolic and membrane-bound states changes from that normally observed, the distribution of precursor maltose-binding protein changes in parallel. These results support the idea that cytosolic SecA plays a role in export. With the aim of determining the roles of the multiple binding sites for ATP on SecA, we compared the export defect in a culture of E. coli expressing a temperature-sensitive allele of secA with the defect in a culture treated with sodium azide. The results indicate that the mutational change and treatment with sodium azide inhibit export by affecting different steps in the cycle of ATP binding and hydrolysis by SecA.
Chun
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Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
Randall
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D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D033902:Periplasmic Binding Proteins; D011498:Protein Precursors / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D019810:Sodium Azide
D018528:ATP-Binding Cassette Transporters; D001386:Azides; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C461599:maltose transport system, E coli; D000255:Adenosine Triphosphate; D019810:Sodium Azide; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins