Endocytosis, intracellular transport and transcytosis of the toxic protein ricin by a polarized epithelium

Abstract

The toxic plant protein ricin binds to both the apical and basolateral surface domains of MDCK (strain I) cells grown on polycarbonate filters. Endocytosis of 125I-labeled ricin was not only higher from the basolateral than from the apical surface--an observation which can be explained by the higher surface area of the basolateral surface--but it also appeared to be more efficient when measured as a percentage of total cell-associated ricin. Monovalent ricin-horseradish peroxidase (Ri-HRP), which is known to behave like native ricin with respect to intracellular transport, also binds to, and is taken up from, both the apical and the basolateral surfaces. Initially, after 10 to 15 min, molecules taken up from the two surface domains at 37 degrees C are present in two separate (basolateral and apical) early endosomal populations. This can also be obtained by incubating for 60 min at 18 degrees C. However, after 30 to 60 min at 37 degrees C, most internalized ligand is found in apical lysosomes, regardless from which surface endocytosis took place. Experiments with endocytosis of cationized ferritin from the apical pole and HRP or Ri-HRP from the basolateral pole showed that intermixing in apical lysosomes (or prelysosomes) of molecules taken up from the two poles occurs. Bidirectional transcytosis involving coated pits of both 125I-labeled ricin and Ri-HRP was demonstrated and was found to be most efficient (as measured in per cent of endocytosed toxin) from the apical pole. Transcytosis was strongly reduced at 18 degrees C, and no transepithelial transport of ricin could be measured at 4 degrees C. Transcytosed ricin was intact and could intoxicate new cells. Finally, delivery of ricin internalized from both the apical and the basolateral surface to the apically localized trans-Golgi network occurred at 37 degrees C but not at 18 degrees C, and ricin inhibited protein synthesis largely with the same kinetics following uptake from the two poles. Incubation at 18 degrees C strongly inhibited the toxic effect of ricin. These data show that ricin can intoxicate epithelia from both sides and also penetrate tight epithelial barriers in intact form.