FtsW acts directly and is sufficient to mediate lipid II translocation.
FtsW is an essential protein that localizes to the site of cell division and contains ten predicted transmembrane segments. Interestingly, the gene encoding FtsW is always located next to that encoding MurG, the enzyme responsible for producing lipid II at the cytoplasmic face of the membrane, prompting the authors to investigate whether FtsW plays a part in lipid II translocation. The authors used a fluorescence resonance energy transfer (FRET)-based assay in which cell-derived vesicles containing an NBD-labelled lipid II donor in the inner leaflet of the membrane were incubated with a membrane-impermeable TMR-labelled vancomycin acceptor, so that FRET occurred only after lipid II translocation. In vesicles derived from an Escherichia coli strain that overexpressed FtsW, the authors observed more translocation of lipid II than in vesicles derived from wild-type cells. Conversely, in vesicles prepared from an E. coli strain in which FtsW had been depleted, they observed a decrease in lipid II translocation. To test whether the involvement of FtsW in this process was direct, the authors reconstituted large unilamellar vesicles (LUVs) containing NBD–lipid II with or without purified FtsW. In the LUVs lacking FtsW, the addition of dithionate to reduce the NBD label resulted in quenching of 50% of the fluorescent signal, which corresponds to the lipid II present in the outer membrane leaflet. In LUVs containing FtsW, this quenching increased to 70%, indicating that FtsW acts directly and is sufficient to mediate lipid II translocation.
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