Cysteine-directed cross-linking demonstrates that helix 3 of SecE is close to helix 2 of SecY and helix 3 of a neighboring SecE.
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.
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
A
A
Kaufmann
E H
EH
Manting
A K
AK
Veenendaal
A J
AJ
Driessen
C
C
van der Does
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D029968:Escherichia coli Proteins; D007930:Leucine / Q000235:genetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids / Q000138:chemical synthesis; D017433:Protein Structure, Secondary; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C088616:SecG protein, E coli; C057841:SecY protein, E coli; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; D007930:Leucine; D003545:Cysteine