Nuclear localization of the Escherichia coli cytolethal distending toxin CdtB subunit.
Cytolethal distending toxin (CDT) is a heterotrimeric protein toxin produced by several bacterial pathogens. Cells exposed to CDT die from either activation of the mitotic checkpoint cascade or apoptosis. Introduction of the purified CdtB subunit, a homologue of mammalian type I DNase, into cells mimics the action of the CDT holotoxin. Mutant CdtBs lacking DNase activity are devoid of biological activity. Chromosomal DNA appears to be the CDT target; thus, nuclear translocation of CdtB must precede cytolethal activity. Examination of the CdtB sequence indicates the presence of putative candidate bipartite nuclear localization signals (NLS). Here, we examine the functionality of the two potential NLS sequences found in the Escherichia coli CdtB-II. Nuclear translocation of EcCdtB-II was examined by monitoring the localization of an EcCdtB-II-EGFP fusion in Cos-7 cells. Our results indicated that EGFP-EcCdtB-II localized to the nucleus. The candidate EcCdtB-II-II NLS sequences were modified by site-directed mutagenesis such that tandem arginine residues were changed to threonine and serine respectively. Mutation of both putative NLS sequences had no effect on EcCdtB-II-associated DNase activity; however, cell cycle arrest and nuclear localization were significantly impaired in cells that received CDT reconstituted from the EcCdtB-II-DeltaNLS mutants. When HeLa cells were electroporated with the EcCdtB-II-DeltaNLS1 and the EcCdtB-II-NLS double mutants, toxicity was not observed, whereas the activity of EcCdtB-II-DeltaNLS2 was similar to that of wild-type EcCdtB-II. These data indicate that the putative NLS sequences are important for CDT-mediated action arrest and that they are likely to function in the nuclear translocation of EcCdtB-II.
McSweeney
Leslie A
LA
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Rm 018 Biological Science Building, 5007 Rockhill Road, Kansas City, MO 64110, USA.
Dreyfus
Lawrence A
LA
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002453:Cell Cycle / Q000502:physiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C067044:cytolethal distending toxin