Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA.
The translocon is a membrane-embedded protein assembly that catalyzes protein movement across membranes. The core translocon, the SecYEG complex, forms oligomers, but the protein-conducting channel is at the center of the monomer. Defining the properties of the SecYEG protomer is thus crucial to understand the underlying function of oligomerization. We report here the reconstitution of a single SecYEG complex into nano-scale lipid bilayers, termed Nanodiscs. These water-soluble particles allow one to probe the interactions of the SecYEG complex with its cytosolic partner, the SecA dimer, in a membrane-like environment. The results show that the SecYEG complex triggers dissociation of the SecA dimer, associates only with the SecA monomer and suffices to (pre)-activate the SecA ATPase. Acidic lipids surrounding the SecYEG complex also contribute to the binding affinity and activation of SecA, whereas mutations in the largest cytosolic loop of the SecY subunit, known to abolish the translocation reaction, disrupt both the binding and activation of SecA. Altogether, the results define the fundamental contribution of the SecYEG protomer in the translocation subreactions and illustrate the power of nanoscale lipid bilayers in analyzing the dynamics occurring at the membrane.
Alami
Meriem
M
Department of Biochemistry and Molecular Biology, Life Sciences Institute, Faculty of Medicine, University of British Columbia, British Columbia, Canada.
Dalal
Kush
K
Lelj-Garolla
Barbara
B
Sligar
Stephen G
SG
Duong
Franck
F
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D008051:Lipid Bilayers / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D049329:Nanostructures; D011485:Protein Binding; D021381:Protein Transport / Q000502:physiology; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins