ISOLATION OF FUNCTIONAL, COATED, ENDOCYTIC VESICLES

Brief internalization of [I-125]transferrin was used to label coated endocytic vesicles, which were then purified using a combination of (H2O)-H-2 and (H2O)-H-2/Ficoll density gradients. Purification was monitored using an assay measuring fusion of endocytic organelles, so as to isolate functional vesicles. Isolated vesicles had all the properties of clathrin-coated vesicles, being enriched for the major components of clathrin coats and uncoated by either 1 M Tris-HCl or an uncoating ATPase. Nearly half of the labeled vesicles were able to participate in subsequent fusion events, as measured by the cell-free assay. Fusion was specific, requiring energy and cytosol, and being sensitive to N-ethyl maleimide.