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 125I-tyramine- cellobiose-asialoorosomucoid (125I-TC-AOM) more extensively than that of 125I-tyramine-cellobiose-ricin (125I-TC-ricin), compatible with the notion that the two probes are internalised via different mechanisms. Subcellular fractionation experiments indicated that 125I-TC-ricin entered a denser endocytic organelle than that receiving 125I-TC-AOM. To determine whether the separation of the two probes was due to a different transport kinetics (i.e. that 125I-TC-ricin is transported more rapidly to a later, denser compartment than 125I-TC-AOM) the cells were incubated at 18 degreesC to allow a slower internalisation/transport of the labelled probes. The results obtained showed, again, that the early endosomes containing 125I-TC-ricin were significantly denser than those containing 125I-TC-AOM. We also employed the horseradish peroxidase (HRP)-diaminobenzidine (DAB) density shift technique of Courtoy et al. [6] to determine whether 125I- TC-ricin and 125I-TC-AOM were in separate endosomes early after their uptake. The results showed that early endosomes containing 125I-TC-AOM were density shifted whereas those containing 125I-TC-ricin were unaffected by the density shift procedure. The use of probes labelled with 125I-TC allowed us to identify compartments involved in the degradation of 125I-TC-AOM and 125I-TC-ricin, by measuring acid soluble radioactivities in the gradient fractions. It was found that 125I-TC- ricin was degraded mainly in endosomes, whereas 125I-TC-AOM, as expected, was degraded mainly in lysosomes.