DIRECT MEASUREMENT OF CLATHRIN-COATED VESICLE FORMATION USING A CELL-FREE ASSAY

Factors controlling the last stages of clathrin-coated vesicle formati on were investigated using an assay allowing direct measurement of the detachment of these vesicles from the plasma membrane, Plasma membran es from cultured cells surface-labelled with I-125-alpha(2)-macroglobu lin (a ligand that preferentially associates with clathrin-coated pits ) a ere isolated by sonication of cells attached to a poly-L-lysine-co ated substratum and incubated in the presence of nucleotide(s) +/- cyt osol, A significant proportion of the membrane-associated radioactivit y was released into the incubation medium in sedimentable form (14 x 1 0(6) g). The nucleotide and ligand specificities of this process toget her with the results of a series of biochemical, morphological and gra dient analyses, led to the conclusion that measurement of the released sedimentable radioactivity provides a direct estimate of the formatio n of clathrin-coated vesicles from clathrin-coated pits. A morphologic al analysis of quick-frozen replicas of these membranes indicated that only the last stages of clathrin-coated vesicle formation were studie d in the assay, Taking advantage of this cell-free system, we demonstr ate that membrane-associated cytosolic factors and GTP-binding protein s, noteably dynamin, play a crucial role, Moreover, although these eve nts can occur in the absence of ATP and Ca2+, optimal conditions for t he formation of clathrin-coated vesicles require the presence of ATP, GTP and cytosol.