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.
Highlights
Diphtheria toxin (DT;* 58 kD) is a typical single-chain AB toxin composed of three functional domains: the aminoterminal catalytic (C) domain corresponds to fragment A (21 kD), and the transmembrane (T) and carboxy-terminal receptor-binding domains comprise fragment B (37 kD) of Because DAB389IL-2 binds with greater affinity to its receptor compared with native DT, this fusion protein toxin has proven to be an effective and novel probe for studying internalization of the C-domain by target cells (Williams et al, 1990)
We demonstrate by mass spectrometry (MS) sequence identification and the effect of specific inhibitors that the chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase (TrR-1) are components of a cytosolic translocation factor (CTF) complex that is essential for the translocation and release of C-domain from early endosomes
We demonstrate that the in vitro translocation of the DAB389IL-2 ADP-ribosyltransferase activity across the membrane of early endosomes and its release into the external milieu requires a CTF complex
Summary
Diphtheria toxin (DT;* 58 kD) is a typical single-chain AB toxin composed of three functional domains: the aminoterminal catalytic (C) domain corresponds to fragment A (21 kD), and the transmembrane (T) and carboxy-terminal receptor-binding domains comprise fragment B (37 kD) of Because DAB389IL-2 binds with greater affinity to its receptor compared with native DT, this fusion protein toxin has proven to be an effective and novel probe for studying internalization of the C-domain by target cells (Williams et al, 1990). Much is known about the mechanisms of receptor-binding and receptor-mediated endocytosis of native DT and the DT-related fusion proteins, little is known about the precise molecular mechanisms of C-domain translocation across the endosomal membrane and its release into the cytosol. There are two conflicting hypotheses for translocation of denatured DT C-domain across the early endosomal membrane. Studies using artificial lipid bilayers suggest that the DT T-domain itself exhibits chaperonin-like properties and is solely sufficient to promote C-domain delivery across the bilayer (Oh et al, 1999; Ren et al, 1999). Studies using partially purified early endosomes that were preloaded with toxin suggest that C-domain translocation across the vesicle membrane is dependent on ATP and the presence of cytosolic components which include -COP (Lemichez et al, 1997). Ren et al (1999) and Hammond et al (2002) have shown that the DT T-domain has chaperonin-like properties, it has a significantly greater affinity for other molten globule-like polypeptides compared with its own C-domain
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