title: Membrane translocation assay based on proteolytic cleavage: application to diphtheria toxin T domain.
abstract: The function of diphtheria toxin translocation (T) domain is to transfer the catalytic domain across the endosomal membrane upon acidification. The goal of this study was to develop and apply an in vitro functional assay for T domain activity, suitable for investigation of structure-function relationships of translocation across lipid bilayers of various compositions. Traditionally, T domain activity in vitro is estimated by measuring either conductance in planar lipid bilayers or the release of fluorescent markers from lipid vesicles. While an in vivo cell death assay is the most relevant to physiological function, it cannot be applied to studying the effects of pH or membrane lipid composition on translocation. Here we suggest an assay based on cleavage of the N-terminal part of T domain upon translocation into protease-loaded vesicles. A series of control experiment was used to confirm that cleavage occurs inside the vesicle and not as the result of vesicle disruption. Translocation of the N-terminus of the T domain is shown to require the presence of a critical fraction of anionic lipids, which is consistent with our previous biophysical measurements of insertion. Application of the proposed assay to a series of T domain mutants correlated well with the results of cytotoxicity assay.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, United States.
authors.forename: Mykola V
authors.initials: MV
authors.lastname: Rodnin
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, United States. Electronic address: aladokhin@kumc.edu.
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Ladokhin
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002463:Cell Membrane Permeability; D004167:Diphtheria Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D059748:Proteolysis; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism
chemical_list: D004167:Diphtheria Toxin; D008051:Lipid Bilayers; D008563:Membrane Lipids; D053835:Unilamellar Liposomes; D013917:Thrombin
keywords: Diphtheria toxin; Large unilamellar vesicles; Membrane translocation; Thrombin proteolysis; pH-triggered membrane insertion