Stage-specific assays for coated pit formation and coated vesicle budding in vitro

Internalization of biotin-S-S-¹²⁵I-transferrin (¹²⁵I-BSST) into semiintact A431 cells were assessed by two different criteria which have allowed us to distinguish partial reactions in the complex overall process of receptor-mediated endocytosis. Early events resulting in the sequestration of ligand into deeply invaginated coated pits were measured by inaccessibility of ¹²⁵I-BSST to exogenously added antibodies. Later events involving coated vesicle budding and membrane fission were measured by resistance of ¹²⁵I-BSST to reduction by the membrane impermeant-reducing agent, MesNa. Acquisition of Ab inaccessibility occurred very efficiently in this cell-free system (∼50% of total cell-associated ¹²⁵I-BSST became inaccessible) and could be inhibited by anti-clathrin mAbs and by antibodies directed against the cytoplasmic domain of the transferrin-receptor. In contrast, acquisition of MesNa resistance occurred less efficiently (∼10-20% of total cell-associated ¹²⁵I-BSST) and showed differential sensitivity to inhibition by anti-clathrin and anti-transferrin receptor mAbs. Both partial reactions were stimulated by ATP and cytosol; indicating at least two ATP-requiring events in receptor-mediated endocytosis. The temperature dependence of both reactions was similar to that for ¹²⁵I-BSST internalization in intact cells with no activity being observed below 10deg;C. Morphological studies using gold-labeled ligands confirmed that internalization of transferrin receptors into semiintact A431 cell occurred via coated pits and coated vesicles and resulted in delivery of ligand to endosomal structures.