Mapping the sites of interaction between SecY and SecE by cysteine scanning mutagenesis

In Escherichia coli, the SecYEG complex mediates the translocation and memb rane integration of proteins. Both genetic and biochemical data indicate in teractions of several transmembrane segments (TMSs) of SecY with SecE. By m eans of cysteine scanning mutagenesis, we have identified intermolecular si tes of contact between TMS7 of SecY and TMS3 of SecE. The cross-linking of SecY to SecE demonstrates that these subunits are present in a one-to-one s toichiometry within the SecYEG complex. Sites in TMS3 of SecE involved in S ecE dimerization are confined to a specific a-helical interface and occur i n an oligomeric SecYEG complex. Although crosslinking reversibly inactivate s translocation, the contact between TMS7 of SecY and TMS3 of SecE remains unaltered upon insertion of the preprotein into the translocation channel. These data support a model for an oligomeric translocation channel in which pairs of SecYEG complexes contact each other via SecE.