Receptor-mediated endocytosis, which plays a critical role in many physiologically important processes, occurs at specialized regions of the plasma membrane (PM), called coated pits. At these sites, clathrin and APs assemble into a protein lattice that concentrates receptor-ligand complexes and deforms the membrane. The GTPase dynamin then self-assembles into a collar at the necks of deeply invaginated coated pits and is required to detach a coated vesicle. In addition to these major constituents of the endocytic machinery, several new proteins have been identified based on their interactions with clathrin, APs, or dynamin. However, we do not know which of these components constitutes the minimal essential machinery for vesicle budding, their mechanism of action, or the function of ancillary components in this process. I propose to develop novel assays using highly purified PM fractions that efficiently and faithfully reconstitute each step along the pathway of CCV formation, including AP2, clathrin, and dynamin recruitment, ligand sequestration and internalization, and the release of free coated vesicles. In the long-term, these stage-specific assays will be used to establish the minimal requirements, the hierarchy of molecular interactions, and the mechanisms governing coated vesicle formation.
