Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis.
Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.
D001212:Asialoglycoproteins; D001692:Biological Transport; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D002460:Cell Line; D003102:Colloids; D004705:Endocytosis; D006046:Gold / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D008247:Lysosomes / Q000378:metabolism*; D008854:Microscopy, Electron; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000378:metabolism; D011955:Receptors, Drug / Q000502:physiology*; D014168:Transferrin / Q000378:metabolism*
D001212:Asialoglycoproteins; D003102:Colloids; D009961:Orosomucoid; D011955:Receptors, Drug; D014168:Transferrin; C017030:asialoorosomucoid; D006046:Gold