Signal peptide determinants of SecA binding and stimulation of ATPase activity.
A signal peptide is required for entry of a preprotein into the secretory pathway, but how it functions in concert with the other transport components is unknown. In Escherichia coli, SecA is a key component of the translocation machinery found in the cytoplasm and at membrane translocation sites. Synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and three sets of model signal sequences varying in hydrophobicity and amino-terminal charge were generated. These were used to establish the requirements for interaction with SecA. Binding to SecA, modulation of SecA conformations sensitive to protease, and stimulation of SecA-lipid ATPase activity occur with functional signal sequences but not with transport-incompetent ones. The extent of SecA interaction is directly related to the hydrophobicity of the signal peptide core region. For signal peptides of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge. The results demonstrate unequivocally that the signal peptide, in the absence of the mature protein, interacts with SecA in aqueous solution and in a lipid bilayer. We show a clear parallel between the hierarchy of signal peptide characteristics that promote interaction with SecA in vitro and the hierarchy of those observed for function in vivo.
Wang
L
L
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
Miller
A
A
Kendall
D A
DA
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008081:Liposomes; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D012697:Serine Endopeptidases
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008081:Liposomes; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; D000266:Adenylyl Imidodiphosphate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins