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.

A 10-aa motif in transmembrane helix 1 of diphtheria toxin that is conserved in anthrax edema factor, anthrax lethal factor, and botulinum neurotoxin serotypes A, C, and D was identified by BLAST, CLUSTAL W, and MEME computational analysis. Using the diphtheria toxin-related fusion protein toxin DAB 389IL-2, we demonstrate that introduction of the L221E mutation into a highly conserved residue within this motif results in a nontoxic catalytic domain translocation deficient phenotype. To further probe the function of this motif in the process by which the catalytic domain is delivered from the lumen of early endosomes to the cytosol, we constructed a gene encoding a portion of diphtheria toxin transmembrane helix 1, T1, which carries the motif and is expressed from a CMV promoter. We then isolated stable transfectants of Hut102͞6TG cells that express the T1 peptide, Hut102͞6TG-T1. In contrast to the parental cell line, Hut102͞6TG-T1 cells are ca. 10 4 -fold more resistant to the fusion protein toxin. This resistance is completely reversed by coexpression of small interfering RNA directed against the gene encoding the T1 peptide in Hut102͞ 6TG-T1 cells. We further demonstrate by GST-DT140-271 pull-down experiments in the presence and absence of synthetic T1 peptides the specific binding of coatomer protein complex subunit ␤ to this region of the diphtheria toxin transmembrane domain.early endosomes ͉ translocation ͉ ADP-ribosyltransferase ͉ coatomer protein complex subunit ␤ T he intoxication of eukaryotic cells by diphtheria toxin follows an ordered series of interactions between the toxin and cellular factors that lead to inhibition of protein synthesis and cell death (1). Biochemical, genetic, and x-ray crystallographic analysis of the toxin has shown the protein to be composed of three distinct domains: an N-terminal catalytic (C) domain, a central transmembrane (T) domain, and the C-terminal receptor-binding domain (2-5). The intoxication process is initiated by the binding of the toxin to its cell surface receptor, a heparin binding epidermal growth factor-like precursor, and CD9 (6, 7). Once bound to its receptor, the toxin is internalized by receptormediated endocytosis into an early endosomal compartment (8). Upon acidification of the endosomal lumen by vesicular ATPase, the T domain undergoes a conformational change and spontaneously inserts into the vesicular membrane, forming an 18-to 22-Å pore or channel (9, 10). It is widely believed that the C domain, in a fully denatured form, is specifically thread through this channel and released into the cytosol. Once the C domain is refolded into an active conformation, it catalyzes the NAD ϩ -dependent ADP ribosylation of elongation factor 2 (EF-2), causing irreversible inhibition of protein synthesis and death of the cell by apoptosis (11,12). Although the endosomal membrane translocation of the C domain is understood in broad terms, the precise molecular mechanism(s) of this step in the intoxication process has remained largely unknown.Two hypotheses for C ...

No abstract

Much is known about the structure function relationships of a large number of bacterial protein toxins, the nature of their cell surface receptors, and their enzymatic activities which lead to the inactivation of their respective cytosolic targets. Despite this wealth of knowledge a detailed understanding of the mechanisms which underlie translocation of the catalytic domain across the eukaryotic cell membrane to the cytosol, the penultimate event in the intoxication process, have been slow in developing. In the case of diphtheria toxin, two prominent hypotheses have been advanced to explain how the catalytic domain is translocated from the lumen of endocytic vesicles to the target cell cytosol. We discuss each of these hypotheses and provide an overview of recent observations that tend to favor a mechanism employing a Cytosolic Translocation Factor complex in the entry process. This facilitated mechanism of translocation appears to rely upon protein-protein interactions between conserved domains within the transmembrane domain of diphtheria toxin with host cell factors to effect delivery of the enzymatic moiety. We have recently identified a 10 amino acid motif in the transmembrane domain of diphtheria toxin that is conserved in anthrax Lethal and Edema Factors, as well as in botulinum neurotoxins A, C and D. Stable eukaryotic cell transfectants that express a peptide containing this motif become resistant to the toxin, and sensitivity is completely restored by co-expression of siRNA which inhibits peptide expression. Data obtained from use of the protein fusion toxin DAB(389)IL-2 in cytotoxicity assays using susceptible Hut 102/6TG and resistant transfectant Hut102/6TG-T1 cells, as well as pull down assays have led to the formulation of a working model of facilitated delivery of the diphtheria toxin catalytic domain to the cytosol of target cells which is discussed in detail.