Monitoring the activity of single translocons.
Recent studies introduced a novel view that the SecYEG translocon functions as a monomer and interacts with the dimeric SecA ATPase, which fuels the preprotein translocation reaction. Here, we used nanodisc reconstituted SecYEG to characterize the functional properties of single copies of the translocon. Using a method based on intermolecular Forster resonance energy transfer, we show for the first time that isolated nanodisc reconstituted SecYEG monomers support preprotein translocation. When several copies of SecYEG were co reconstituted within a nanodisc, no change in translocation kinetics was observed, suggesting that SecYEG oligomers do not facilitate enhanced translocation. In contrast, nanodisc reconstituted monomers of the PrlA4 variant of SecYEG showed increased translocation rates. Experiments based on intramolecular Forster resonance energy transfer within the nanodisc isolated monomeric SecYEG demonstrated a nucleotide dependent opening of the channel upon interaction with SecA. In conclusion, the nanodisc reconstituted SecYEG monomers are functional for preprotein translocation and provide a new prospect for single molecule analysis of dynamic aspects of protein translocation.
Journal of molecular biology