title: Stage-specific assays for coated pit formation and coated vesicle budding in vitro.
abstract: 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 (approximately 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 (approximately 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 10 degrees 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.
authors.affiliation: Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
authors.forename: S L
authors.initials: SL
authors.lastname: Schmid
authors.forename: E
authors.initials: E
authors.lastname: Smythe
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000911:Antibodies, Monoclonal; D002474:Cell-Free System; D002966:Clathrin / Q000276:immunology / Q000502:physiology; D003034:Coated Pits, Cell-Membrane / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis; D006801:Humans; D007158:Immunologic Techniques; D008854:Microscopy, Electron; D011990:Receptors, Transferrin / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D013696:Temperature; D014168:Transferrin / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D002966:Clathrin; D011990:Receptors, Transferrin; D014168:Transferrin; D000255:Adenosine Triphosphate