Mapping the sites of interaction between SecY and SecE by cysteine scanning mutagenesis.
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.
Veenendaal
A K
AK
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
van der Does
C
C
Driessen
A J
AJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine; D019281:Dimerization; D004220:Disulfides / Q000032:analysis; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D021381:Protein Transport; D000069816:SEC Translocation Channels
D001426:Bacterial Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C057841:SecY protein, E coli; D003545:Cysteine