RESUMEN
In Escherichia coli, the SecYEG complex mediates the translocation and membrane integration of proteins. Both genetic and biochemical data indicate interactions of several transmembrane segments (TMSs) of SecY with SecE. By means of cysteine scanning mutagenesis, we have identified intermolecular sites 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 stoichiometry within the SecYEG complex. Sites in TMS3 of SecE involved in SecE dimerization are confined to a specific alpha-helical interface and occur in an oligomeric SecYEG complex. Although cross-linking reversibly inactivates 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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cisteína , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Membrana Celular/metabolismo , Dimerización , Disulfuros/análisis , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Subunidades de Proteína , Transporte de Proteínas , Canales de Translocación SECRESUMEN
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 transmembrane segment (TMS) 2 of SecY and TMS 3 of SecE to probe possible sites of interaction between the integral membrane subunits. The SecY and SecE single-Cys mutants were cloned individually and in pairs into a secYEG expression vector and functionally overexpressed. Oxidation of the single-Cys pairs 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 opposite alpha-helical face of TMS 3 of SecE was found to interact with a neighboring SecE molecule. Formation of this SecE dimer did not affect the high-affinity binding of SecA to SecYEG and ATP hydrolysis, but blocked preprotein translocation and thus uncouples the SecA ATPase activity from translocation. Conditions that prevent membrane deinsertion of SecA markedly stimulated the interhelical contact between the SecE molecules. The latter demonstrates a SecA-mediated modulation of the protein translocation channel that is sensed by SecE.