The SecYEG translocon of Escherichia coli mediates the translocation of preproteins across the cytoplasmic membrane. Here, we have examined the role of the proposed lateral gate of the translocon in translocation. A dual cysteine cross-linking approach allowed the introduction of cross-linker arms of various lengths between adjoining aminoacyl positions of transmembrane segments 2b and 7 of the lateral gate. Oxidation and short spacer linkers that fix the gate in the closed state abolished preprotein translocation, whereas long spacer linkers support translocation. The cross-linking data further suggests that SecYEG lateral gate opening and activation of the SecA ATPase are coupled processes. It is concluded that lateral gate opening is a critical step during SecA-dependent protein translocation.
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