title: FtsH-dependent processing of RNase colicins D and E3 means that only the cytotoxic domains are imported into the cytoplasm.
abstract: It has long been suggested that the import of nuclease colicins requires protein processing; however it had never been formally demonstrated. Here we show that two RNase colicins, E3 and D, which appropriate two different translocation machineries to cross the outer membrane (BtuB/Tol and FepA/TonB, respectively), undergo a processing step inside the cell that is essential to their killing action. We have detected the presence of the C-terminal catalytic domains of these colicins in the cytoplasm of target bacteria. The same processed forms were identified in both colicin-sensitive cells and in cells immune to colicin because of the expression of the cognate immunity protein. We demonstrate that the inner membrane protease FtsH is necessary for the processing of colicins D and E3 during their import. We also show that the signal peptidase LepB interacts directly with the central domain of colicin D in vitro and that it is a specific but not a catalytic requirement for in vivo processing of colicin D. The interaction of colicin D with LepB may ensure a stable association with the inner membrane that in turn allows the colicin recognition by FtsH. We have also shown that the outer membrane protease OmpT is responsible for alternative and distinct endoproteolytic cleavages of colicins D and E3 in vitro, presumably reflecting its known role in the bacterial defense against antimicrobial peptides. Even though the OmpT-catalyzed in vitro cleavage also liberates the catalytic domain from colicins D and E3, it is not involved in the processing of nuclease colicins during their import into the cytoplasm.
authors.affiliation: CNRS, UPR 9073, Institut de Biologie Physico-Chimique, 75005 Paris, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Chauleau
authors.affiliation: CNRS, UPR 9073, Institut de Biologie Physico-Chimique, 75005 Paris, France.
authors.forename: Liliana
authors.initials: L
authors.lastname: Mora
authors.affiliation: CNRS, UPR 9073, Institut de Biologie Physico-Chimique, 75005 Paris, France.
authors.forename: Justyna
authors.initials: J
authors.lastname: Serba
authors.affiliation: CNRS, UPR 9073, Institut de Biologie Physico-Chimique, 75005 Paris, France. Electronic address: zamaroczy@ibpc.fr.
authors.forename: Miklos
authors.initials: M
authors.lastname: de Zamaroczy
mesh_terms: D049069:ATP-Dependent Proteases / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003087:Colicins / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology* / Q000235:genetics; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D002352:Carrier Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011956:Receptors, Cell Surface; C488855:ceiC protein, E coli; C501879:colicin D immunity protein, E coli; C029489:enterobactin receptor; C489181:tonB protein, E coli; D012260:Ribonucleases; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli; D012697:Serine Endopeptidases; C026515:type I signal peptidase