title: In vivo trapping of FtsH substrates by label-free quantitative proteomics.
abstract: FtsH is the only membrane-bound and essential protease in Escherichia coli. It is responsible for the degradation of regulatory proteins and enzymes such as the heat-shock sigma factor RpoH or LpxC, the key enzyme of lipopolysaccharide biosynthesis. To find new FtsH targets, we trapped substrates in E. coli cells from exponential and stationary growth phase by using a proteolytically inactive FtsH variant. Subsequent analysis of the isolated FtsH-substrate complexes by label-free nanoLC-MS/MS revealed more than 50 putative FtsH substrates, among them five already known substrates. Four out of thirty-seven tested candidates were found to be novel FtsH substrates as shown by in vivo degradation experiments. Six other candidates were degraded by one or more other protease(s). The FtsH substrates SecD and ExbD are involved in transport processes across the membrane, whereas the physiological roles of YlaC and YhbT are yet unknown. The presence of the previously identified YfgM degron in two of the novel substrates suggests general rules for substrate recognition of this unique protease.
authors.affiliation: Ruhr-Universität Bochum, Lehrstuhl Biologie der Mikroorganismen, Bochum, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Arends
authors.affiliation: Ruhr-Universität Bochum, Medizinisches Proteom-Center, Bochum, Germany.
authors.forename: Nikolas
authors.initials: N
authors.lastname: Thomanek
authors.affiliation: Ruhr-Universität Bochum, Medizinisches Proteom-Center, Bochum, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Kuhlmann
authors.affiliation: Ruhr-Universität Bochum, Medizinisches Proteom-Center, Bochum, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Marcus
authors.affiliation: Ruhr-Universität Bochum, Lehrstuhl Biologie der Mikroorganismen, Bochum, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Narberhaus
mesh_terms: D049069:ATP-Dependent Proteases / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli
keywords: AAA protein; ATP-dependent protease; Escherichia coli; FtsH; Microbiology; Proteolysis