Intracellular transport of asialoglycoproteins in rat hepatocytes. Evidence for two subpopulations of lysosomes.
The intracellular transport and degradation of asialoorosomucoid (AOM) in isolated rat hepatocytes was studied by means of subcellular fractionation in Nycodenz gradients. The asialoglycoprotein was labelled by covalent attachment of a radioiodinated tyramine-cellobiose adduct ( [125I]TC) which leads to labelled degradation products being trapped intracellularly and thus serving as markers for the degradative organelles. The ligand was initially (1 min) in a slowly sedimenting (small) vesicle and subsequently in larger endosomes. Acid-soluble, radioactive degradation products were first found in a relatively light lysosome whose distribution coincided in the gradient with that of the larger endosome. Later (30 min) degradation products were found in denser lysosomes which banded in the same region of the gradient as the lysosomal enzyme, beta-acetylglucosaminidase. Colchicine, monensin and leupeptin all inhibited degradation of [125I]tyramine-cellobiose asialoorosomucoid ( [125I]TC-AOM) and reduced the formation of degradation products in both the light and the dense lysosomes. In presence of monensin and colchicine no undegraded ligand was seen in the dense lysosome, suggesting that uptake in these vesicles was inhibited. Leupeptin allowed accumulation of undegraded ligand in the dense lysosome. Therefore, transfer from light to dense lysosomes is not dependent on degradation as such. In the presence of monensin two peaks of undegraded ligand were found in the gradients. It seems possible that in the monensin-sensitive endosomes, dissociation of the ligand-receptor complex is inhibited, allowing ligand to recycle with the receptors in small vesicles.
D000818:Animals; D001212:Asialoglycoproteins; D002458:Cell Fractionation; D002475:Cellobiose; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D003864:Depression, Chemical; D007976:Leupeptins / Q000494:pharmacology; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008247:Lysosomes / Q000145:classification / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008985:Monensin / Q000494:pharmacology; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014439:Tyramine
D001212:Asialoglycoproteins; D007976:Leupeptins; D009961:Orosomucoid; C017030:asialoorosomucoid; C038394:tyramine-cellobiose adduct; D002475:Cellobiose; D008985:Monensin; C032854:leupeptin; D003078:Colchicine; D014439:Tyramine