Endocytosis from Coated Pits of Shiga Toxin: A Glycolipid-binding Protein from Shigella dysenteriae

Evidence is presented that endocytosis is involved in the transport to the cytosol of the cytotoxin from Shigella dysenteriae 1, Shiga toxin, which acts by removal of a single adenine residue in 28-S ribosomal RNA. Inhibition of endocytosis by ATP depletion of the cells prevented toxin uptake. Exposure of HeLa $3 and Vero cells to toxin at low extracellular pH, where translocation to the cytosol, but not endocytosis is inhibited, allowed the toxin to accumulate in a compartment where it was protected against antibodies to the toxin. Upon transfer of the cells to normal medium endocytosed toxin entered the cytosol. Electron microscopical studies of cells exposed at 0°C to a toxin-horseradish peroxidase (HRP) conjugate, or to unconjugated toxin followed by horse antitoxin antibodies and then protein G-gold, revealed that the Shiga toxin binding sites were randomly distributed on the cell surface, without any preference to, for example, coated pits. In contrast, when cells were exposed to toxin at 37°C, the binding sites were preferentially localized in coated pits. The Shiga-HRP conjugate was also seen in endosomes, lysosomes, and in the Golgi region. Endocytosis by the coated pit/coated vesicle pathway was selectively inhibited by acidification of the cytosol. Under these conditions, both the uptake of toxin-HRP conjugates and intoxication of the cells were inhibited. Evidence from the literature as well as our own results suggest that Shiga toxin binding sites are glycolipids. Thus, Shiga toxin appears to be the first example of a lipid-binding ligand that is endocytosed from coated pits. URING recent years endocytic uptake of a large number of ligands has been studied. Most of these ligands are bound to protein receptors, and they are usually taken up by the coated pit/coated vesicle pathway (5, 13, 15, 21, 22, 32, 34). However, there has been an ongoing discussion as to whether alternative pathways of endocytosis also exist. Thus, ultrastructural data indicated that the glycolipid-binding toxins, cholera toxin and tetanus toxin (24, 46), as well as monoclonal antibodies directed against a cell surface glycolipid (45) are endocytosed from uncoated areas of the membrane. Furthermore, fluid-phase endocytosis appeared to continue even when receptor-mediated endocytosis was inhibited by high osmolarity in the medium (4). Also, the toxic protein ricin, which binds not only to glycoproteins, but also to glycolipids with terminal galactose residues (for review, see reference 28), continued to be taken up when endocytosis from coated pits was blocked by potassium depletion of the cells (25), or by acidification of the cytosol (37, 38). Similar results were obtained with human rhinovirus 2 (20). Also insulin, which binds to protein receptors, has been reported to be endocytosed from uncoated areas of the membrane in some cell types (11, 12, 42), suggesting that the alternative pathway of endocytosis is not limited to ligands binding to glycolipids. Shiga toxin is an extremely toxic protein produced by Shigella dysenteriae 1, which is considered the most virulent agent of bacillary dysentery (8, 27, 51). Shiga-like toxins produced by Escherichia coli are associated with hemorrhagic colitis and hemolytic uremic syndrome (17, 26). Shiga toxin consists of an enzymatically active A-chain and a pentameric B-subunit that binds to glycolipids containing the Gala 1-4 Gal sequence (3, 16, 18, 19). The A-chain penetrates to the cytosol and inactivates the 60-S ribosomal subunits (33) by depurination of a single adenosine residue in 28-S RNA (9). It has been assumed that endocytosis is involved in the penetration of the toxin to the cytosol (28, 35). In the present paper we present evidence that this is the case and that the endocytosis occurs by the coated pit/coated vesicle pathway despite the glycolipid nature of the Shiga toxin receptor. Materials and Methods