Cysteine-directed cross-linking demonstrates that helix 3 of SecE is closeto helix 2 of SecY and helix 3 of a neighboring SecE

Preprotein translocation in Escherichia coli is mediated by translocase, a multimeric membrane protein complex with SecA as the peripheral ATPase and SecYEG as the translocation pore. Unique cysteines were introduced into tra nsmembrane segment (TMS) 2 of SecY and TMS 3 of SecE to probe possible site s of interaction between the integral membrane subunits. The SecY and SecE single-Cys mutants were cloned individually and in pairs into a secYEG expr ession vector and functionally overexpressed. Oxidation of the single-Cys p airs revealed periodic contacts between SecY and SecE that are confined to a specific alpha-helical face of TMS 2 and 3, respectively. A Cys at the op posite alpha-helical face of TMS 3 of SecE was found to interact with a nei ghboring SecE molecule. Formation of this SecE dimer did not affect the hig h-affinity binding of SecA to SecYEG and ATP hydrolysis, but blocked prepro tein translocation and thus uncouples the SecA ATPase activity from translo cation. Conditions that prevent membrane deinsertion of SecA markedly stimu lated the interhelical contact between the SecE molecules. The latter demon strates a SecA-mediated modulation of the protein translocation channel tha t is sensed by SecE.