Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes.

Abstract

Abstract. 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 Rvesicles (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. CIRCULATING 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)(48)(49)54). After internalization of Abbreviations used in this paper." ASGP, asialoglycoprotein; ASGP-R, asialoglycoprotein ...

Acidification of morphologically distinct endosomes in mutant and wild-type Chinese hamster ovary cells.

“…6) could concentrate the recycling receptors in a low volume/high surface area compartment and thus exclude luminal ligand (6,7). The late, prelysosomal endosomes is the last organelle before lysosomes, and lacks recycling elements and would correspond to receptor-negative endosomes (22).…”

Receptor-mediated endocytosis of testicular transferrin by germinal cells of the rat testis

“…The results however also revealed that resialation was incomplete and followed very slow kinetics (Regoeczi et al , 1982). Al though there seems to be ev idence for the existence of both pathways (Hopkins, 1983b;Yarnashiro et al, 1984;Mueller and Hubbard, 1986), there also secms to be strong indications that the TRG associated pùthwùy is relatively minor (Sibille et al, 1986: Ajioka et al, 1986.…”

“…For example, half-times of 3-5 minutes have been calculated for the release of apoTf from celis such as rabbit reticulocytes (Iocopetta and Morgan, 1981), chinese hamster ovary (CHO) cells (Schrnid et al, 1988) and hepatocytGS (Ciechanover et al , 1983b (Schmid et al, 1988;Mueller and Hubbard, 1986;Hopkins and Trowbridge, 1983).…”

Dissection of the multifunctional “receptor-recycling” endocytic compartment of hepatocytes

“…44,45 Despite the morphological characterization, the attempts to isolate those structures enriched in recycling-receptors, from liver homogenates, using subcellular fractionation and the subsequent biochemical analysis, are limited. 19,23,24,27,46 When highly purified endosomes from rat liver 27 were used for a comprehensive biochemical and electron microscopic characterization, 28,29,33,47 the morphological and pleiotropic functional complexity of the RRC became evident and, although RRC was first characterized as the endosomal fraction enriched in recycling receptors (transferrin receptor, asialoglycoprotein receptor, LDL receptor-related protein, and LDL receptor), the confirmation that contains molecules that follow the transcytotic pathway such as pIgR-pIgA complexes 48 as well as coated-like vesicles enriched with caveolin-1, 29,49 prompted us to introduce an operational ''functional'' dissection of RRC into 3 subcompartments: RRCr, to those structures predominantly devoted to recycling of receptors; RRCt, to structures involved in transporting molecules to the bile (transcytosis); and RRCc, for those structures enriched in caveolin-1.…”

“…A number of methods have been developed for the isolation of hepatic endosomes. 13,[19][20][21][22][23][24][25][26][27] In general, subcellular fractionation explores the differences in the equilibrium densities between organelles or the electric charges of membrane fractions (free-flow electrophoresis). 13,19 However, the similarity in the densities of endocytic vesicles, Golgi membranes, and other smooth membranes has led to the development of methods for specifically modifying the density of endocytic vesicles based on their ligand content.…”