Diphtheria toxin does not enter resistant cells by receptor-mediated endocytosis.

Diphtheria toxin (DT) and pseudomonas toxin are two distinct microbial toxins which inhibit protein synthesis in an identical manner, i.e., by the inactivation of cytoplasmic elongation factor 2. Although murine cells bind both toxins, they are 10,000-fold less sensitive to DT than to pseudomonas toxin. This suggests that the level of resistance resides at some event after binding. We have previously shown that pseudomonas toxin enters mouse LM fibroblasts by receptor-mediated endocytosis, a process in which ligand is internalized via specialized clathrin-coated pits and moves to the Golgi region and then to the lysosomes. Here, we visually follow the entry and trafficking of DT by resistant mouse fibroblasts. A biotinyl-toxin-avidin-gold system was used to visualize DT on the ultrastructural level. DT entered resistant cells through non-clathrin-coated regions of the plasma membrane and within 2.5 to 5 min was seen in lysosomes. Only rarely was DT seen in coated pits or in the Golgi region. Furthermore, the temperature dependence of internalization of 125I-labeled horseradish peroxidase and 125I-labeled DT by LM cells was similar. On the basis of these observations, we postulate that DT does not enter DT-resistant LM cells by receptor-mediated endocytosis and that receptor-mediated endocytosis is required for efficient expression of toxicity.