title: Binding of SecA to the SecYEG complex accelerates the rate of nucleotide exchange on SecA.
abstract: SecYEG translocase mediates the transport of preproteins across the inner membrane of Escherichia coli. SecA binds the membrane-embedded SecYEG protein-conducting channel with high affinity and then drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the kinetics of nucleotide binding to SecA while associated with the SecYEG complex. Lipid-bound SecA was separated from Se-cYEG-bound SecA by sedimentation of the proteoliposomes through a glycerol cushion, which maintains the SecA native state and effectively removes the lipid-bound SecA fraction. Nucleotide binding was assessed by means of fluorescence resonance energy transfer using fluorescent ATP analogues as acceptors of the intrinsic SecA tryptophan fluorescence in the presence of a tryptophanless variant of the SecYEG complex. Binding of SecA to the SecYEG complex elevated the rate of nucleotide exchange at SecA independently of the presence of preprotein. This defines a novel pretranslocation activated state of SecA that is primed for ATP hydrolysis upon preprotein interaction.
authors.affiliation: Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9750 AA Haren, The Netherlands.
authors.forename: Paolo
authors.initials: P
authors.lastname: Natale
authors.forename: Jelto
authors.initials: J
authors.lastname: Swaving
authors.forename: Chris
authors.initials: C
authors.lastname: van der Does
authors.forename: Jeanine
authors.initials: J
authors.lastname: de Keyzer
authors.forename: Arnold J M
authors.initials: AJ
authors.lastname: Driessen
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D004735:Energy Transfer; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C088616:SecG protein, E coli; C057841:SecY protein, E coli; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins