Screening for stabilization of proteins with a trans-translation signature in Escherichia coli selects for inactivation of the ClpXP protease.
Trans-translation is a process that adds a hydrophobic peptide tag to the C-terminus of polypeptides, which causes them to become unstable. We designed a genetic screen to identify factors involved in the degradation of trans-translated products, using the green fluorescent protein (GFP) fused to the trans-translation tag as a reporter. Two screens were devised to identify insertional mutants that stabilize such substrates. Only disruption of the clpX or clpP gene resulted in stabilization of the tagged substrates. The sspB gene product was recently shown to be a specificity-enhancing factor for the ClpXP degradation machine. In the wild-type background, targeted inactivation of the sspB gene failed to stabilize the tagged substrate. These results indicate that the ATP-dependent ClpXP protease is probably the only main component involved in the degradation of cytoplasmic trans-translated proteins in Escherichia coli that can be completely inactivated.
Bohn
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Laboratoire des Réseaux de Régulations, C.N.R.S./U.M.R. 8621, Institut de Génétique et Microbiologie, Centre Scientifique d'Orsay, Université Paris-Sud, Bâtiment 400, 91405 Orsay Cedex, France.
Binet
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Bouloc
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D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D049071:Endopeptidase Clp; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D017930:Genes, Reporter; D015698:Genomic Library; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics*; D014170:Transformation, Genetic
D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012697:Serine Endopeptidases; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases