Binding of SecA to the SecYEG complex accelerates the rate of nucleotide exchange on SecA.
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.
Natale
Paolo
P
Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9750 AA Haren, The Netherlands.
Swaving
Jelto
J
van der Does
Chris
C
de Keyzer
Jeanine
J
Driessen
Arnold J M
AJ
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
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