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Monitoring the Activity of Single Translocons

Taufik, I., Kedrov, A., Exterkate, M. & Driessen, A. J. M., 15-Nov-2013, In : Journal of Molecular Biology. 425, 22, p. 4145-4153 9 p.

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  • Monitoring the Activity of Single Translocons

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DOI

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.

Original languageEnglish
Pages (from-to)4145-4153
Number of pages9
JournalJournal of Molecular Biology
Volume425
Issue number22
Publication statusPublished - 15-Nov-2013

    Keywords

  • 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, CHANNEL, ATPASE

ID: 6004918