Demonstration of a specific Escherichia coli SecY-signal peptide interaction.
Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The signal peptide-SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA.
Wang
Ligong
L
Department of Molecular and Cell Biology, The University of Connecticut, Storrs, Connecticut 06269, USA.
Miller
Alexander
A
Rusch
Sharyn L
SL
Kendall
Debra A
DA
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D026901:Membrane Transport Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; D010649:Phenylalanine; D001710:Biotin; C053764:benzoylphenylalanine; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins