Synthetic signal peptides specifically recognize SecA and stimulate ATPase activity in the absence of preprotein.
Although it is known that virtually all exported proteins require a signal peptide, it is not clearly understood how the signal peptide interfaces with the translocation machinery to achieve transport. In this study we document a direct interaction between the signal peptide and SecA, a primary component of the translocase in Escherichia coli, and show that the signal peptide itself can stimulate SecA-lipid ATPase activity. Using synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and two model sequences, we find that the extent of stimulation of SecA ATPase activity by the different peptides parallels the hierarchy of results found for in vivo function (Izard, J. W., Doughty, M. B., and Kendall, D. A. (1995) Biochemistry 34, 9904-9912). The peptide-induced activity requires a lipid to protein molar ratio of at least 300:1 and liposomes enriched in negatively charged phospholipids. Furthermore, specific binding of the signal peptide to SecA was demonstrated using chemical cross-linking and competition with unlabeled peptides.
Miller
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A
Department of Molecular and Cell Biology, The University of Connecticut, Storrs, Connecticut 06269, USA.
Wang
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L
Kendall
D A
DA
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport; D002474:Cell-Free System; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000378:metabolism*; D011510:Proteolipids; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D011510:Proteolipids; D000069816:SEC Translocation Channels; C020485:proteoliposomes; D000469:Alkaline Phosphatase; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins