Isolation of an endocytic compartment from A431 cells using a density modification procedure employing a receptor-specific monoclonal antibody complexed with colloidal gold.
Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endocytosis in human epidermoid carcinoma (A431) cells. The isolation protocol involves density modification of endosome elements in A431 cells, caused by the receptor-dependent binding and internalization at 20 degrees C of colloidal gold-transferrin receptor antibody (B3/25) particles. The use of 125I-labelled gold-B3/25 provides a radioactive marker for the endosome compartment, the major peak being recovered at the bottom of a continuous sucrose gradient at a density of 1.23g ml-1. Enzyme markers characteristic of other cytoplasmic compartments are present only in negligible amounts in this fraction and L-[35S]methionine-labelling of the cells indicates approximately a 200-fold enrichment of 125I-labelled gold-B3/25 versus protein. Electron microscopy of the endosome-rich fraction reveals that we have isolated a highly purified population of small gold-containing vesicles and tubules from which the transferrin receptor can be immunoprecipitated using the B3/25 antibody. Gel electrophoresis and fluorography of L-[35S]-methionine-labelled cells suggests that these elements contain a characteristic profile of approximately 10 major proteins of which three appear to be specifically enriched. In cells incubated with [125I]transferrin, 12% of the ligand sediments with the gold-labelled elements. We conclude, therefore, that the components we have isolated play a role in the intracellular processing of the transferrin-transferrin receptor complexes.
D000911:Antibodies, Monoclonal; D002294:Carcinoma, Squamous Cell / Q000032:analysis; D002458:Cell Fractionation; D004705:Endocytosis; D011992:Endosomes / Q000032:analysis*; D006048:Gold Colloid, Radioactive; D006801:Humans; D011990:Receptors, Transferrin / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D006048:Gold Colloid, Radioactive; D011990:Receptors, Transferrin