title: The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex.
abstract: In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650-800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.
authors.affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA. ratts@bu.edu
authors.forename: Ryan
authors.initials: R
authors.lastname: Ratts
authors.forename: Huiyan
authors.initials: H
authors.lastname: Zeng
authors.forename: Eric A
authors.initials: EA
authors.lastname: Berg
authors.forename: Clare
authors.initials: C
authors.lastname: Blue
authors.forename: Mark E
authors.initials: ME
authors.lastname: McComb
authors.forename: Cathy E
authors.initials: CE
authors.lastname: Costello
authors.forename: Johanna C
authors.initials: JC
authors.lastname: vanderSpek
authors.forename: John R
authors.initials: JR
authors.lastname: Murphy
mesh_terms: D016227:Benzoquinones; D020134:Catalytic Domain; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D047029:Lactams, Macrocyclic; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D013058:Mass Spectrometry; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology; D013601:T-Lymphocytes / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D015003:Yeasts
chemical_list: D016227:Benzoquinones; D004167:Diphtheria Toxin; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D007783:Lactones; D018942:Macrolides; D020652:Peptide Elongation Factor 2; D011809:Quinones; D013880:Thioredoxin-Disulfide Reductase; C035359:monorden; C001277:geldanamycin