Location of Repository

Monitoring the Activity of Single Translocons

By Intan Taufik, Alexej Kedrov, Marten Exterkate and Arnold J. M. Driessen

Abstract

<p>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 PrIA4 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. (C) 2013 Elsevier Ltd. All rights reserved.</p>

Topics: protein transport, secretion; protein oligomerization; protein dynamics; membrane proteins; BACTERIAL CYTOPLASMIC MEMBRANE; PHOSPHOLIPID-BILAYER NANODISCS; PROTEIN-TRANSLOCATION; ESCHERICHIA-COLI; SIGNAL SEQUENCE; PREPROTEIN TRANSLOCATION; PRECURSOR PROTEINS; SECY COMPLEX;
Year: 2013
DOI identifier: 10.1016/j.jmb.2013.08.012
OAI identifier: oai:pure.rug.nl:publications/b8b06fa9-62f8-4938-b88b-6c938a041d23
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://dx.doi.org/10.1016/j.jm... (external link)
  • Suggested articles


    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.