title: Monitoring the activity of single translocons.
abstract: 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 Förster 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 Förster 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.
authors: Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors: Intan
authors: I
authors: Taufik
authors: Alexej
authors: A
authors: Kedrov
authors: Marten
authors: M
authors: Exterkate
authors: Arnold J M
authors: AJ
authors: Driessen
mesh_terms: D003902:Detergents; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D021381:Protein Transport; D000069816:SEC Translocation Channels; D012996:Solutions
chemical_list: D003902:Detergents; D029968:Escherichia coli Proteins; D000069816:SEC Translocation Channels; D012996:Solutions
keywords: DDM; FCCS; FCS; FRET; Förster resonance energy transfer; SEC; fluorescence correlation spectroscopy; fluorescence cross-correlation spectroscopy; membrane proteins; n-dodecyl β-d-maltoside; protein dynamics; protein oligomerization; protein transport; secretion; size-exclusion chromatography