Assembly of Clathrin-Coated Pits onto Purified Plasma Membranes

• Published in: Science (American Association for the Advancement of Science) 1987-05, Vol.236 (4801), p.558-563
• Main Authors: Moore, Mary Shannon, Mahaffey, David T., Brodsky, Frances M., Richard G. W. Anderson
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - pharmacology; Analysis; Antibodies; Biological and medical sciences; Biology; Botany; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cell membranes; Cell physiology; Cell research; Clathrin; Clathrin - metabolism; Coated Pits, Cell-Membrane - drug effects; Coated Pits, Cell-Membrane - physiology; Coated Pits, Cell-Membrane - ultrastructure; Coated vesicles; Cultured cells; Cytological research; Cytoplasm; Endocytosis; Endosomes - physiology; Fibroblasts; Fundamental and applied biological sciences. Psychology; Gels; Humans; Kinetics; Membrane Proteins - metabolism; Microscopy, Electron; Molecular and cellular biology; Molecular Weight; Physiological aspects; Plasma membranes; Proteins; Temperature
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.2883727

During receptor-mediated endocytosis, coated pits invaginate to form coated vesicles, clathrin and associated proteins dissociate from the vesicle membrane, and these proteins form new coated pits at the cell surface. As a means of elucidating molecular mechanisms thag govern the function of coated pits, the assembly phase of this cycle was reconstituted by incubating purified membranes that were treated to remove endogenous coated pits with cytoplasm extracted from cultured cells. The in vitro assembly of coated pits on these membranes satisfactorily mimics many features of coated pit formation in the intact cell. These studies indicate that: (i) the membranes contain a limited number of coated pit assembly sites that bind clathrin with high affinity; (ii) the half-time for assembly is 5 minutes both at 4°C and 37°C; (iii) during assembly, proteins with molecular sizes of 180, 110, and 36 kilodaltons are recruited to the plasma membrane; and (iv) assembly is not dependent on adenosine triphosphate, but this nucleotide triggers a temperature-dependent loss of coated pits that are assembled in the absence of adenosine triphosphate.