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.

Bacterial protein toxins and lipids: pore formation or toxin entry into cells

“…Indeed, a depletion of membrane sphingolipids facilitates DT transport (Spilsberg et al, 2005). In addition, cytosolic factors, which include β-COP (β-subunit of coatamer protein I), Hsp90 (heat-shock protein 90), thioredoxin reductase and ATP, are required for DT fragment A translocation across a vesicle membrane (Lemichez et al, 1997;Ratts et al, 2003). The interchain disulfide bridge is reduced during the translocation process, and the A chain refolds at the neutral pH found in the cytosol.…”

Topography of the Hydrophilic Helices of Membrane-Inserted Diphtheria Toxin T Domain: TH1−TH3 as a Hydrophilic Tether

“…However, exactly how T domain assists translocation is unclear. Furthermore, translocation may be aided by cytoplasmic factors (25,26) Defining the structure of the membrane-inserted T domain is likely to provide insights into the mechanism of A chain translocation. The T domain is primarily comprised of 9 α-helices (TH1-9).…”

Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel

“…The cells then were washed and incubated for an additional 2 h on ice with wild-type or mutant LF N that had been labeled with [ 35 S]methionine in an in vitro transcription͞translation system (Promega). The cells then were washed with ice-cold PBS at pH 5.0 or 8.0, incubated at 37°C for 1 min, and either treated with Pronase to digest residual untranslocated 35 S at the cell surface or left untreated as controls. The cells then were lysed, and 35 S liberated into the lysis buffer was assayed.…”

“…The cells then were washed with ice-cold PBS at pH 5.0 or 8.0, incubated at 37°C for 1 min, and either treated with Pronase to digest residual untranslocated 35 S at the cell surface or left untreated as controls. The cells then were lysed, and 35 S liberated into the lysis buffer was assayed. The percent translocation was defined as dpm protected from Pronase͞dpm bound to cells ϫ 100.…”

“…PA binding to the cell surface and translocation of 35 S-LF N were assayed as described in refs. 13 and 15.…”