Isolation and characterization of a highly enriched preparation of receptosomes (endosomes) from a human cell line.

Abstract

Receptor-mediated endocytosis proceeds by transfer of receptor-ligand complexes from clathrin-coated pits at the cell surface to uncoated endocytic vesicles termed receptosomes (or endosomes). These vesicles have now been purified more than 37-fold based on their content of newly internalized epidermal growth factor. 125I-labeled EGF was bound to human KB carcinoma cells at 4 degrees C, and then the cells were warmed to 37 degrees C for 8 min and disrupted. The purification scheme involved density gradient centrifugation on colloidal silica and sucrose and gel filtration on Sephacryl S-1000. Relative to homogenate, receptosomes are enriched 4.3-fold in their cholesterol content and depleted in enzyme markers for plasma membranes (2- to 3-fold) and lysosomes (9-fold). Receptosomes have a polypeptide composition that is different from plasma membrane, lysosome, and other homogenate fractions. They are enriched in transferrin receptors (30-fold) and in unidentified Mr 70,000-75,000 glycoprotein(s); they contain phosphomannosyl receptors. They do not contain detectable amounts of clathrin.