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Published 31 March 2003. doi:10.1083/jcb.200210028
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© The Rockefeller University Press, 0021-9525/2003/3/1139 $5.00
The Journal of Cell Biology, Volume 160, Number 7, 1139-1150

Article

The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex



Ryan Ratts1,2,3, Huiyan Zeng2, Eric A. Berg1,4, Clare Blue2, Mark E. McComb1,4, Cathy E. Costello1,3,4, Johanna C. vanderSpek2 and John R. Murphy2,5

1 Department of Biochemistry, Section of Molecular Medicine
2 Department of Medicine, Section of Molecular Medicine
3 Cell and Molecular Biology Program, Boston University School of Medicine, Boston, MA 02118
4 Mass Spectrometry Resource, Boston University School of Medicine, Boston, MA 02118
5 Department of Microbiology, Boston University School of Medicine, Boston, MA 02118

Address correspondence to Ryan Ratts, 650 Albany Street, EBRC 830, Boston University School of Medicine, Boston, MA 02118. Tel.: (617) 638-6062. Fax: (617) 638-6020. E-mail: ratts{at}bu.edu; or John R. Murphy, 650 Albany Street, EBRC 830, Boston University School of Medicine, Boston, MA 02118. Tel: (617) 638-6014. Fax: (617) 638-6020. E-mail: jmurphy{at}medicine.bu.edu

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.

Key Words: endosome; Hsp 90; thioredoxin reductase; geldanamycin; radicicol

The online version of this article includes supplemental material.

H. Zeng's present address is Dept. of Pathology, Harvard Medical School, Boston, MA 02115.

C. Blue's present address is Division of Infection and Immunity, University of Glasgow, Glasgow G12 8QQ, UK.

* Abbreviations used in this paper: br, bovine recombinant; C, catalytic; CTF, cytosolic translocation factor; DT, diphtheria toxin; EF-2, elongation factor 2; ESI, electrospray ionization; hr, human recombinant; Hsp, heat shock protein; IL-2, interleukin-2; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; T, transmembrane; TrR-1, thioredoxin reductase.


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