Abstract. The passage of pulse doses of asialoglycoproteins through the endosomal compartments of rat liver hepatocytes was studied by subcellular fractionation and EM. The kinetics of disappearance of radiolabeled asialofetuin from light endosomes prepared on Ficoll gradients were the same as the kinetics of disappearance of asialoorosomucoid-horse radish peroxidase reaction products from intracellular membrane-bound structures in the blood sinusoidal regions of hcpatocytes. The light endosomes were therefore identifiable as being derived from the peripheral early endosomc compartment. In contrast, the labeling of dense endosomes from the middle of the Ficoll gradient correlated with EM showing large numbers of reaction product-containing structures in the nonsinusoidal parts of the hepatocytc.In ceil-free, postmitochondrial supcrnatants, we have previously observed that dense cndosomes, but not light endosomcs, interact with lysosomcs. Cell-free interaction between isolated dense endosomes and lysosomes has now been reconstituted and analyzed in three ways: by transfer of radiolabclcd ligand from endosomal to lysosomal densities, by a fluorescence dequenching assay which can indicate membrane fusion, and by measurement of content mixing. Maximum transfer of radiolabel to lysosomal densities required ATP and GTP plus cytosolic components, including N-cthylmaleimide-sensitive factor(s). Dense endosomes incubated in the absence of added lysosomcs did not mature into vesicles of lysosomal density. Content mixing, and hence fusion, between endosomes and lysosomcs was maximal in the presence of cytosol and ATP and also showed inhibition by N-ethylmaleimide. Thus, we have demonstrated that a fusion step is involved in the transfer of radiolabcled ligand from an isolated endosome fraction derived from the nonsinusoidal regions of the hepatocyte to preexisting lysosomcs in a ceU-frcc system. U NDERSTANDING of vesicular traffic pathways in the cell and their control has increased markedly in recent years, very largely as a result of the development of cell-free systems which permit identification of the proteins required. Components which operate at many steps on the secretory pathway have thus been characterized and shown to include GTP-binding proteins (Novick and Brcnnwald, 1993), N-ethylmaleimide (NEMy-sensitive factor, its soluble attachment proteins and, most recently, receptor proteins which confer specificity between vesicle and target (Sollner et al., 1993; lhkizawa and Malhotra, 1993 1. Abbreviations used in this paper: AMP-PNP, 5'-adenylylimidodiphosphate; ASE asialofetuin; ASOR-HRP, asialoorosomucoid-horse radish peroxidase conjugate; bplgA, biotinylated polymeric IgA; NEM, N-ethylmaleimide; RI8, octadecylrhodamine/~-chloride; RIPA, 1% sodium, 1% Triton X-100, 0.2% NP-40, 0.15 M NaCI, 0.05 M Tris, pH 7.5; STM, 0.25 M sucrose, 10 mM N-tris 01ydroxymethyl)-methyl-2-aminoethane sulphonic acid and I mM Mg 2+ (pH 7.4). Smythe and Warren, 1991; Schmid, 1993). In vivo it is known that hepatocytes internalize ligand...

1. A gamma camera was used to monitor continuously the uptake of radiolabelled polymeric immunoglobulin A (pIgA) into the rat body after intravenous injection. Uptake into liver was fast but, since the peak of liver labelling occurred only after 9-15 min, it was not sufficiently rapid to constitute a pulse dose. A perfused, isolated rat liver system was therefore established which could be given a single pass dose of pIgA; a variety of tests showed such livers remained viable for at least 3 h and could be subsequently fractionated on Ficoll and Nycodenz gradients with normal distributions of marker enzymes. 2. Subcellular fractionation at different times after a single pass dose of pIgA showed that whilst pIgA appeared sequentially in sinusoidal plasma membrane, light endosomes, dense endosomes, very dense endosomes and lysosomes as in vivo, the predominance of pIgA in the light endosome compartment disappeared much earlier than after injection in vivo of pIgA, presumably because this compartment was not being continuously loaded over the first 10-15 min. The time course of appearance of label in bile was unchanged. A large excess of unlabelled asialofetuin did not change these patterns, indicating that the asialoglycoprotein receptor was not involved. 3. Low doses of the microtubule agent colchicine reduced the proportion of pIgA reaching the bile, but subcellular fractionation of treated liver showed that distribution of label amongst liver fractions was little changed, although the overall liver pIgA content had increased. This would suggest that pIgA did not remain in the common compartment which could have supplied bile or lysosomes but rather flowed out of it as rapidly as in untreated liver but towards those compartments supplying the lysosomes. 4. Experiments with nocodazole, which reversibly disrupts microtubules, showed that very little of the pIgA taken into an inhibited liver appeared in the bile after nocodazole was removed 30 min later, even though a second dose of pIgA, given after nocodazole removal, appeared in bile with a normal time course. The first dose of pIgA must therefore have passed beyond the compartments competent to supply the bile before nocodazole was removed. Such compartments were undamaged since the second dose of pIgA appeared in bile normally. We therefore conclude that the bulk of pIgA must be supplied to the bile from light or dense endosomes rather than from very dense endosomes and lysosomes.

A monoclonal antibody raised against human colostrum secretory component produced even staining of hepatocyte plasma membranes, as well as bile duct lining cells, in all sections examined from eight normal and three abnormal human livers. Human bile samples incubated with free secretory component degraded it to varying extents, probably proteolytically; true levels of free secretory component will therefore often be higher than those reported. It seems likely that human liver resembles that of other mammals in transferring polymeric IgA through hepatocytes to the bile by means of the polymeric IgA receptor.

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