Endocytosed ricin and asialoorosomucoid follow different intracellular pathways in hepatocytes

Earlier studies have suggested that fluid phase endocytosis in rat hepatocytes takes place via a clathrin-independent mechanism [1,2]. This observation suggests that a relatively large amount of plasma membrane outside coated pits may be involved in hepatic endocytosis. Ricin, which binds to galactose residues on glycoproteins and glycolipids, has, in this report, been used as a general marker for the plasma membrane of hepatocytes, The endocytosis of ricin was compared with that of asialoorosomucoid (AOM) which is taken up exclusively via clathrin-coated pits, Hypertonic medium has been shown to inhibit uptake via coated pits more effectively than clathrin-independent uptake [3-5]. It was found, in this study, that the addition of 100 mM sucrose to the incubation medium inhibited the uptake of I-125-tyramine-cellobiose-asialoorosomucoid (I-125-TC-AOM) more extensively than that of I-125-tyramine-cellobiose-ricin (I-125-TC-ricin), compatible with the notion that the two probes are internalised via different mechanisms. Subcellular fractionation experiments indicated that I-125-TC-ricin entered a denser endocytic organelle than that receiving I-125-TC-AOM. To determine whether the separation of the two probes was due to a different transport kinetics (i,e, that I-125-TC-ricin is transported more rapidly to a later, denser compartment than I-125-TC-AOM) the cells were incubated at 18 degrees C to allow a slower internalisation/transport of the labelled probes. The results obtained showed, again, that the early endosomes containing I-125-TC-ricin were significantly denser than those containing I-125-TC-AOM. We also employed the horseradish peroxidase (HRP)-diaminobenzidine (DAB) density shift technique of Courtoy et al. [6] to determine whether I-125-TC-ricin and I-125-TC-AOM were in separate endosomes early after their uptake. The results showed that early endosomes containing I-125-TC-AOM were density shifted whereas those containing I-125-TC-ricin were unaffected by the density shift procedure. The use of probes labelled with I-125-TC allowed us to identify compartments involved in the degradation of I-125-TC-AOM and I-125-TC-ricin, by measuring acid soluble radioactivities in the gradient fractions. It was found that I-125-TC-ricin was degraded mainly in endosomes, whereas I-125-TC-AOM, as expected, was degraded mainly in lysosomes. (C) 1998 Elsevier Science B.V. All rights reserved.