Antibodies that block the ligand binding site of the cation‐dependent mannose 6‐phosphate specific receptor (Mr 46,000 MPR) were used to probe the function of the receptor in transport of lysosomal enzymes. Addition of the antibodies to the medium of Morris hepatoma 7777 cells, which express only the Mr 46,000 MPR, resulted in a decreased intracellular retention and increased secretion of newly synthesized lysosomal enzymes. In fibroblasts and HepG2 cells that express the cation‐independent mannose 6‐phosphate specific receptor (Mr 215,000 MPR) in addition to the Mr 46,000 MPR, antibodies against the Mr 46,000 MPR inhibited the intracellular retention of newly synthesized lysosomal enzymes only when added to the medium together with antibodies against the Mr 215,000 MPR. Morris hepatoma (M.H.) 7777 did not endocytose lysosomal enzymes, while U937 monocytes, which express both types of MPR, internalized lysosomal enzymes. The uptake was inhibited by antibodies against the Mr 215,000 MPR, but not by antibodies against the Mr 46,000 MPR. These observations suggest that Mr 46,000 MPR mediates transport of endogenous but not endocytosis of exogenous lysosomal enzymes. Internalization of receptor antibodies indicated that the failure to mediate endocytosis of lysosomal enzymes is due to an inability of surface Mr 46,000 MPR to bind ligands rather than its exclusion from the plasma membrane or from internalization.

We studied the intracellular transport of secretory and membrane proteins in the human hepatoma cell line HepG-2 infected with vesicular stomatitis virus. Cells were pulselabeled in the presence of [3SS]methionine and chased in the presence of the lysosomotropic agent primaquine. At a concentration of 0.3 mM primaquine effectively inhibited the secretion of albumin and, to a lesser extent, that of orosomucoid and transferrin. The drug also prevented the budding of virus particles at the cell surface. The intracellular transport to the Golgi complex of the membrane protein VSV-G was not affected by primaquine as it acquires resistance to endo-/~-N-acetylglucosaminidase H at the same rate as in control cells. Addition of primaquine at various times after the initiation of the chase period indicates that the effect of primaquine occurs just before secretion. In confirmation of the biochemical data, immunocytochemical localization of albumin in cells treated with NH4CI demonstrated that albumin accumulated in vesicles at the trans side of the Golgi complex. The effect of primaquine on secretion was also compared with its effect on receptor recycling. The dose-response characteristics of the effect of primaquine on receptor recycling are identical to those of the effects on protein secretion and virus budding. These results indicate that both processes involve the same transport mechanism, and/or that they occur via at least one identical intracellular compartment.Secretory and plasma membrane proteins are transported from the rough endoplasmic reticulum (RER) ~ via the Golgi complex to the cell exterior. The transport rate between the RER and the Golgi complex differs for individual proteins and varies between 20 and 120 min. In rat and human hepatoma cells infected with vesicular stomatitis virus (VSV), albumin and the viral membrane protein VSV-G pass through the Golgi complex within 20 rain after synthesis, whereas transferrin remains in the RER for almost 2 h before acquisition of the complex sugar configuration (29). After passage through the Golgi complex secretory and membrane (glyco)proteins rapidly (within a few minutes) reach the plasma membrane.Abbreviations used in this paper: ASGP, asialoglycoprotein; ASOR, asialoorosomucoid; endo H, endo-a-N-acetylglucosaminidase; RER, rough endoplasmic reticulum; VSV, vesicular stomatitis virus.THE JOURNAL OF CELL BIOLOGY -VOLUME 101 AUGUST 1985 531-539 © The Rockefeller University Press • 0021-9525185/08/0531/09 $1.00Another intracellular pathway in which proteins are specifically targetted along specific routes is that of receptor-mediated endocytosis of extracellular ligands. The occurrence of receptor recycling has been inferred from studies of a variety of receptor-ligand systems in many different cells, i.e., the receptor-mediated systems for delivery of low density lipoprorein, transferrin, lysosomal enzymes, and asialoglycoproteins (ASGPs) in hepatocytes. During receptor-mediated endocytosis, a ligand binds to a specific cell surface receptor, and the...

Abstract. The receptor for asialoglycoproteins (ASGPR) was localized in human hepatoma Hep G2 cells by means of quantitative immunoelectron microscopy. Without ligand added to the culture medium, we found 34 % of the total cellular receptors on the plasma membrane, 37 % in compartment of uncoupling receptor and ligand (CURL), and 21% in a trans-Golgi reticulum (TGR) that was defined by the presence of albumin after immuno-double labeling. A small percent of the ASGPR was associated with coated pits, the Golgi stacks, and lysosomes. After incubation of the cells with saturating concentrations of the ligand asialo-orosomucoid (ASOR), the number of cell surface receptors decreased to 20% of total cellular receptors, whereas the receptor content of CURL increased by a corresponding amount to 50%. The ASGPR content of TGR remained constant. In contrast, after treatment of the cells with 300 ~tM of the weak base primaquine (PMQ), cell surface ASGPR had decreased dramatically to only 4% of total cellular receptors whereas label in the TGR had increased to 42 %. ASGPR labeling of CURL increased only to 47 %. The labeling of other organelles remained unchanged. This affect of PMQ was independent of the presence of additional ASOR. Implications for the intracellular pathway of the ASGPR are discussed.IVER parenchymal cells possess cell surface receptors that function in the clearance of galactose-terminated glycoproteins from the circulation (1, 17). In addition, these asialoglycoprotein receptors (ASGPR) 1 are present in abundance on the human hepatoma cell line Hep G2 that contains •225,000 high affinity ligand-binding sites per cell (22,23). In Hep G2 cells not exposed to ligand a considerable fraction of the functional ligand-binding sites is located on the cell surface (21). However, the presence of ligand (e.g., asialoorosomucoid [ASOR]) in the extracellular fluid promotes a relative increase in the fraction of receptors located intracellularly (4, 23).Lysosomotropic amines, which accumulate within intracellular acidic organelles (5) thereby neutralizing the internal pH (18, 33), alter the intraceUular distribution of several receptor species. The number of surface-binding sites for LDL (2), mannose-(29) and mannose-6-phosphate-(13) terminated ligands, alpha-2 macroglobulin (15), and asialoglycoproteins (21, 28) is markedly reduced after incubation with these agents. This reduction was seen in both the presence and absence of added ligand. However, in several cases the 1. Abbreviations used in this paper: ASGPR, asialoglycoprotein receptor; ASOR, asialo-orosomucoid; CURL, compartment of uncoupling receptor and ligand; MVB, multi-vesicular body; PMQ, primaquine; TGR, transGolgi reticulum. effect was enhanced in the presence ofligand (2,15,21). This loss of cell surface-binding sites is reversible since after removal of the agent the cell surface ligand-binding capacity is rapidly and fully restored (21, 29). These observations suggest that the receptors accumulate intracellularly in a nonlysosomal compartment. The ai...

The biosynthesis, glycosylation and subcellular localization of the neutral endopeptidase-24.11 were studied in cultured human fibroblasts. The enzyme was synthesized as a precursor (Mr 88,000) containing four or five N-linked oligosaccharides. Within 1 h the synthesis-mature (Mr 94,000) endopeptidase-24.11 was formed and contained sialylated oligosaccharides. The half-life of endopeptidase-24.11 was 3.7 days and in the presence of 10 mM-NH4Cl it increased to 6 days. Mature endopeptidase-24.11 was solubilized with 0.2% saponin and partitioned into Triton X-114. In intact fibroblasts, endopeptidase-24.11 was accessible to antibodies and to neuraminidase even when the treatment was performed at 4 degrees C. The localization of endopeptidase-24.11 to the plasma membrane in cultured fibroblasts was further demonstrated by immunocytochemistry.

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