The Entry of Diphtheria Toxin into the Mammalian Cell Cytoplasm : Evidence for Lysosomal Involvement ROCKFORD

Lysosomotropic amines, such as ammonium chloride, are known to protect cells from the cytotoxic effects of diphtheria toxin. These drugs are believed to inhibit the transport of the toxin from a receptor at the cell exterior into the cytoplasm where a fragment of the toxin arrests protein synthesis. We studied the effects of lysosomotropic agents on the cytotoxic process to better understand how the toxin enters the cytoplasm. The cytotoxic effects of diphtheria toxin were not inhibited by antitoxin when cells were preincu-bated at 37°C with toxin and ammonium chloride, exposed to antitoxin at 4° C, washed to relieve the ammonium chloride inhibition, and finally warmed to 37°C. The antigenic determinants of the toxin were, therefore, either altered or sheltered. It is likely that the combination of ammonium chloride and a low temperature trapped the toxin in an intracellular vesicle from which the toxin could proceed to the cytoplasm. Because lysosomotropic amines raise the pH within acidic intracellular vesicles, such as lysosomes, they could trap the toxin within such a vesicle if an acidic environment were necessary for the toxin to penetrate into the cytoplasm. We simulated acidic conditions which the toxin might encounter by exposing cells with toxin bound to their surface to acidic medium. We then measured the effects of lysosomotropic amines on the activity of the toxin to see if the acidic environment substituted for the function normally inhibited by the drugs. The drugs no longer protected the cells. This suggests that exposing the toxin to an acidic environment, such as that found within lysosomes, is an important step in the penetration of diphtheria toxin into the cytoplasm. Diphtheria toxin is a prokaryotic protein (63,000 mol wt) that arrests protein synthesis in most mammalian cells. The first step in this process occurs at the cell surface where the toxin binds to a receptor. The last step occurs in the cytoplasm where fragment A (21,000 mol wt) of the toxin inactivates elongation factor 2 (EF-2) by catalyzing the covalent attachment of the adenosine diphosphate ribose portion of NAD+ to EF-2 (see reference 2 and 21 for review). The mechanism by which fragment A is transported from the cell exterior to the cyto-plasm is unknown. Theories to explain the transport process suggest either that the toxin forms a pore in the plasma membrane through which fragment A passes (1) or that the toxin is first endocytosed and then somehow …