Measurement of Receptor-mediated Endocytosis of [ u ~ I ] Transferrin , [ u ~ IIEGF , and lmI ] Ricin

Acidification of the cytosol of a number of different cell lines strongly reduced the endocytic uptake of transferrin and epidermal growth factor. The number of transferrin binding sites at the cell surface was increased in acidified cells. Electron microscopic studies showed that the number of coated pits at the cell surface was not reduced in cells with acidified cytosol. Experiments with transferrin-horseradish peroxidase conjugates and a monoclonal anti-transferrin receptor antibody demonstrated that transfen'in receptors were present in ,x,75 % of the coated pits both in control cells and in cells with acidified cytosol. The data therefore indicate that the reason for the reduced endoeytic uptake of transferrin at internal pH <6.5 is an inhibition of the pinching off of coated vesicles. In contrast, acidification of the cytosol had only little effect on the uptake of ricin and the fluid phase marker lucifer yellow. Ricin endocytosed by cells with acidified cytosol exhibited full toxic effect on the cells. Although the pathway of this uptake in acidified cells remains uncertain, some coated pits may still be involved. However, the data are also consistent with the possibility that an alternative endocytic pathway involving smooth (uncoated) pits exists. number of different transport proteins, hormones, growth factors, toxins, and viruses enter cells by receptor-mediated endocytosis. Coated pits and coated vesicles are generally assumed to be involved in this process (for reviews, see references 33, 38, and 59). Upon entry, the ligands are transferred to endosomes where the low pH is essential for dissociation of ligands from their receptors, for release of Fe 3+ from transferrin, and for entry into the cytosol of viruses and certain toxins. In studies of endocytosis and its role in the mechanism of action of many ligands, it is a problem that no specific inhibitor of endocytosis is available. Although treatments with metabolic inhibitors and low temperature inhibit endocytosis, these conditions inhibit most other physiological processes in the cells as well. Recently, removal of cytoplasmic potassium was found to inhibit endocytosis of low density lipoprotein, epidermal growth factor (EGF),~ and transferrin (29, 31, 37). However, such treatment does not efficiently inhibit the formation of coated vesicles in all cell types (37, 44). Furthermore, K + depletion induces cell shrinkage and inhibits anion transport (32). Alternative methods to inhibit endocytosis from coated pits are therefore warranted. In the course of our studies of the mechanism of action of diphtheria toxin, ricin, and related toxic proteins with intracellular sites of action, we noted that acidification of the cytosol inhibited endocytic uptake of different ligands to different extents (46). Here we report that acidification of the 1. Abbreviations used in this paper: DMO, ['442]dimethyloxazolidine 2, 4-dione; EGF, epidermal growth factor; HRP, horseradish peroxidase; Ri-Au, ricin-gold; Tf-Au, transferrin-gold; Tf-HRP, monovalent transferrin-HRP. cytosol efficiently blocks endocytosis of transferrin and EGE whereas the uptake of the toxic plant protein ricin and of the membrane impermeant fluorescent dye, lucifer yellow, is only slightly reduced. Materials and Methods