Abstract
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.