title: Real time observation of single membrane protein insertion events by the Escherichia coli insertase YidC.
abstract: Membrane protein translocation and insertion is a central issue in biology. Here we focus on a minimal system, the membrane insertase YidC of Escherichia coli that inserts small proteins into the cytoplasmic membrane. In a reconstituted system individual insertion processes were followed by single-pair fluorescence resonance energy transfer (FRET), with a pair of fluorophores on YidC and the substrate Pf3 coat protein. After addition of N-terminally labeled Pf3 coat protein a close contact to YidC at its cytoplasmic label was observed. This allowed to monitor the translocation of the N-terminal domain of Pf3 coat protein across the membrane in real time. Translocation occurred within milliseconds as the label on the N-terminal domain rapidly approached the fluorophore on the periplasmic domain of YidC at the trans side of the membrane. After the close contact, the two fluorophores separated, reflecting the release of the translocated Pf3 coat protein from YidC into the membrane bilayer. When the Pf3 coat protein was labeled C-terminally, no translocation of the label was observed although efficient binding to the cytoplasmic positions of YidC occurred.
authors.affiliation: Institute of Microbiology and Molecular Biology, University of Hohenheim, Stuttgart, Germany.
authors.forename: Sophie
authors.initials: S
authors.lastname: Winterfeld
authors.forename: Stefan
authors.initials: S
authors.lastname: Ernst
authors.forename: Michael
authors.initials: M
authors.lastname: Börsch
authors.forename: Uwe
authors.initials: U
authors.lastname: Gerken
authors.forename: Andreas
authors.initials: A
authors.lastname: Kuhn
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D009154:Mutation; D011485:Protein Binding
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C412443:YIDC protein, E coli