COPI coatomer complex proteins facilitate the translocation of anthrax lethal factor across vesicular membranes in vitro

Abstract

The delivery of the diphtheria toxin catalytic domain (DTA) from acidified endosomes into the cytoplasm of eukaryotic cells requires protein-protein interactions between the toxin and a cytosolic translocation factor (CTF) complex. A conserved peptide motif, T1, within the DT transmembrane helix 1 mediates these interactions. Because the T1 motif is also present in the N-terminal segments of lethal factor (LF) and edema factor (EF) in anthrax toxin, we asked whether LF entry into the cell might also be facilitated by target cell cytosolic proteins. In this study, we have used LFnDTA and its associated ADP-ribosyltransferase activity (DTA) to determine the requirements for LF translocation from the lumen of endosomal vesicles to the external medium in vitro. Although low-level release of LFnDTA from enriched endosomal vesicles occurs in the absence of added factors, translocation was enhanced by the addition of cytosolic proteins and ATP to the reaction mixture. We show by GST-LFn pull-down assays that LFn specifically interacts with at least -COP and ␤-COP of the COPI coatomer complex. Immunodepletion of COPI coatomer complex and associated proteins from cytosolic extracts blocks in vitro LFnDTA translocation. Translocation may be reconstituted by the addition of partially purified bovine COPI to the translocation assay mixture. Taken together, these data suggest that the delivery of LF to the cytosol requires either COPI coatomer complex or a COPI subcomplex for translocation from the endosomal lumen. This facilitated delivery appears to use a mechanism that is analogous to that of DT entry. diphtheria toxin ͉ anthrax toxin R eceptor-mediated endocytosis of many bacterial protein toxins ensures their passage through an acidified endosomal compartment. Diphtheria toxin (DT), anthrax toxin (AT), and the botulinum neurotoxins all require exposure to a low-pH environment for the delivery of their respective catalytic domains to the cytosol (1-3). In the case of DT, exposure to a low-pH environment is known to trigger a dynamic change in the transmembrane domain resulting in its spontaneous insertion into the vesicle membrane and the formation of a pore or channel (1). Ratts et al. (4) have demonstrated that the in vitro translocation of the DT catalytic domain from the lumen of partially purified endosomal vesicles through this pore to the external milieu requires the addition of ATP and a cytosolic translocation factor (CTF) complex to the in vitro translocation assay mixture (4). Although the CTF complex has not been fully characterized, several components including Hsp90, thioredoxin reductase, and ␤-COP from the COPI coatomer complex have been shown to be essential components in the catalytic domain delivery process (4-6).Subsequently, Ratts et al. (5) described a putative translocation motif, T1, in DT transmembrane helix 1, which is conserved in anthrax lethal factor (LF), edema factor (EF), and botulinum neurotoxins A, C1, and D (5). Because the L221E mutation in the T1 motif in the diphtheria toxin-related...

Diphtheria Toxin and Cytosolic Translocation Factors

“…The LFn-DTa is a fusion protein consisting of the LF binding domain for PA and the C domain of diphtheria. Tamayo et al (2008) clearly demonstrated that the anthrax LFn-DTa fusion protein construct was translocated across the endosomal membrane in an ATP and cytosol dependent fashion, and this observation was confirmed by Dmochewitz et al (2011). Tamayo et al (2008) also demonstrated using GST-LFn pull downs that the T1 motif in anthrax lethal factor directly binds -COP, as well as zeta ( )-COP, and that a synthetic peptide containing the DT T1 motif blocked this interaction.…”

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

“…Indeed, it has been shown that for several toxins (e.g., diphtheria toxin, anthrax lethal factor, anthrax edema factor), the entry process is facilitated in vivo by specific interactions with dedicated cytosolic factors, such as the coatomer I complex, Hsp90, or thioredoxin reductase, which recognize specific amino acid sequences within the toxin polypeptide chains (44)(45)(46)(47). These interactions likely contribute to pull the catalytic chains of these toxins through the membrane through a Brownian ratchet-like mechanism (48), and could aid toxin refolding within the cytosol.…”

CCT chaperonin complex is required for efficient delivery of anthrax toxin into the cytosol of host cells

“…We have previously shown that LF N unfolding in the early endosome (pH 6.5), but not the late endosome (pH 5), is facilitated by the endosomal chaperone GRP78 (8). Furthermore, translocation of LF N -DTA from isolated endosomes requires the COPI coatamer complex, in addition to other, previously unidentified, cytoplasmic factors (9). The related AB toxins diphtheria and Clostridium botulinum C2 toxin share a requirement with LT for the COPI complex; however, unlike LT, diphtheria, C. botulinum C2 toxin, Clostridium difficile transferase, and Clostridium perfringens iotatoxin also require heat shock protein 90 (Hsp90) and cyclophilin A (26)(27)(28)(29)(30).…”

“…However, we have previously shown that the endosomal chaperone GRP78 (78 kDa glucose regulated protein) facilitates LF N unfolding in the mildly acidic early endosome (pH 6.5), but not the late endosome (pH 5) (8). Furthermore, translocation of the fusion protein LF N -DTA (LFN fused to diphtheria toxin fragment A) from endosomes in vitro requires cytosolic factors, including the coatomer protein complex I (COPI) (9). Thus, host factors clearly play an important role in toxin translocation, reflecting remarkable coevolution of a pathogen and the host.…”