Receptor-mediated Endocytosis of Asialoglycoproteins by Rat Hepatocytes: Receptor-positive and Receptor-negative Endosomes

We have used combinations of subcellular fractionation, specific cytochemical tracers, and quantitative immunoadsorption to determine when, where, and in which intracellular structure internalized asialoglycoproteins (ASGPs) are segregated from their receptor. All membrane vesicles containing the receptor (R+ vesicles) were quantitatively immunoadsorbed from crude microsomes with Staphylococcus aureus cells and affinity-purified anti-ASGP receptor. Using this assay, we varied the time and temperature of exposure of perfused livers to 125I-asialoorosomucoid (125I-ASOR) and followed the movement of ligand from R+ to R vesicles. After 2.5 min at 37°C, 98% of the internalized ligand could be immunoadsorbed and thus was in R+ vesicles. Over the next 12 min of continuous 37°C perfusion with 125I-ASOR, an increasing fraction of the ligand was not immunoadsorbed and therefore was present in R vesicles. A maximum of 30% of the ligand could be found in R vesicles (14-44 min). When livers were maintained at 16"C, ligand was internalized but remained in R+ vesicles. Furthermore, ligand accumulating in R vesicles at 37"C remained there when livers were cooled to 16*C. R endosomes could be separated from R+ endosomes by flotation on sucrose density gradients and visualized by the presence of sequestered ASORhorseradish peroxidase (ASOR-HRP). These structures resembled those labeled by ASOR-HRP in situ: R+ vesicles were relatively dense (1.12 g/cc), frequently tubular or spherical and small (100-nm diam), corresponding to the peripheral and internal tubular endosomes; R structures were of lower density (1.09 g/cc), large (400-nm diam), and resembled internal multivesicular endosomes (MVEs). Endocytosed ASOR-HRP was found in both the peripheral and internal tubular endosomes in situ under conditions where 95% of the ligand was present in R+ vesicles by immunoadsorption, whereas MVEs containing ASOR-HRP were predominant in situ when ligand was found in R vesicles and were often in continuity with the tubular internal endosomes. All of these results suggest that complete segregation of ligand and receptor occurs after arrival in the Golgi-lysosome region of the hepatocyte and that MVEs are R and represent the final prelysosomal compartment. C IRCULATING asialoglycoproteins (ASGPs) t are endocytosed by rat liver parenchymal cells (hepatocytes) and degraded in lysosomes. The ligand pathway has been mapped using electron microscopic (EM) tracers, and several prelysosomal compartments have been identified and biochemically characterized (l l, 12, 18, 19, 52, 53). Mapping the receptor pathway has been more difficult, due, in part, to the existence of internal pools of ASGP receptors (ASGP-R) whose precise locations are still not resolved (12, 37, 52, 56). Each receptor is believed to cycle between the cell surface and the internal pools, mediating the entry of >200 ligand molecules in its lifetime (39, 47-49, 54). After internalization of Abbreviations used in this paper." ASGP, asialoglycoprotein; ASGP-R, asialoglycoprotein receptor; ASOR, asialoorosomucoid; BSA-PBS, 1% bovine serum albumin in 0.02 M NaPi, pH 7.4, 0.15 M NaCI; DAB, diaminobenzidine; EM, electron microscopy; HRP, horseradish peroxidase; MVEs, multivesicular endosomes; R , receptor-negative; R+, receptor-positive; S/I, 0.25 M sucrose in 3 mM imidazole, pH 7.4. the ligand-receptor complex (2, 55), a minimum of two steps would seem to be required for accomplishing both delivery of ligand to lysosomes and delivery of receptor back to the cell surface--dissociation of ligand from receptor and physical separation of the two molecules into distinct intracellular compartments. Much evidence implicates the complex, prelysosomal membrane system collectively termed endosomes as the site(s) for these two steps (e.g., 2, 12, 16, 17, 34, 50, 53, 55, 57). Recently, we identified and biochemically characterized three endosomal compartments of the ASGP pathway in rat hepatocytes (52). The internal (latent) ASGP receptors we detected were in a membrane compartment whose biochemical characteristics were strikingly similar to those of the peripheral and internal tubular endosomes. Results from immunoelectron microscopy have found intracellular ligand and the ASGP receptor together (12), but the precise location as well as the dynamics of the two components remain unclear. In the present study, we have applied subeellular fraction© The Rockefeller University Press, 0021-9525/86/03/0932/11 $1.00 932 The Journal of Cell Biology, Volume 102, March 1986 932-942 on O cber 0, 2017 jcb.rress.org D ow nladed fom ation and quantitative immunoadsorption, together with in situ and in vitro cytochemistry of a specific ASGP tracer, to address the questions of when, where, and in what structures segregation of ligand and receptor occurs. We report here that asialoorosomucoid (ASOR) internalized at 37"C is first found in receptor-positive (R+) endosomes but then appears in receptor-negative (R-) endosomes before entry into lysosornes. Several lines of evidence suggest that this step occurs after transport of ligand into the internal endosomal compartment. Finally, we have found that the R endosomes are multivesicular. Materials and Methods