SecY-SecY and SecY-SecG contacts revealed by site-specific crosslinking.
Protein translocation across the cytoplasmic membrane of Escherichia coli is mediated by the integral membrane complex SecYEG and the peripherally bound ATPase SecA. To probe the environment of the cytoplasmic domains of SecY within the SecYEG complex, we introduced single cysteine residues in each of the six cytoplasmic domains. Neighbouring SecY molecules with a single cysteine residue in cytoplasmic domains C1, C2 or C6 formed a disulfide bond upon oxidation. The presence of the disulfide bond between two C2 domains reversibly inhibited protein translocation. Chemical crosslinking showed that the C2 and C3 domains are in close proximity of SecG and chemical modification of the cysteine residue in the C5 domain with N-ethyl-maleimide or fluorescein-5-maleimide inactivates the SecYEG complex. Taken together, our data give novel insights in the interactions between subunits of the SecYEG complex and emphasise the importance of cytoplasmic domain C5 for SecY functioning.
van der Sluis
Eli O
EO
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands.
Nouwen
Nico
N
Driessen
Arnold J M
AJ
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005033:Ethylmaleimide / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011498:Protein Precursors / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D000069816:SEC Translocation Channels
D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D029968:Escherichia coli Proteins; D005452:Fluoresceins; D008565:Membrane Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C088616:SecG protein, E coli; C057841:SecY protein, E coli; C057184:outer membrane protein A precursor (E coli); C037041:fluorescein 5-maleimide; D003545:Cysteine; D005033:Ethylmaleimide