Direct demonstration of the endocytic function of caveolae by a cell-free assay

The endocytic function of caveolae was challenged by taking advantage of a cell-free assay directly measuring the detachment of receptor-containing ve sicles from isolated plasma membranes. Plasma membranes from cultured cells surface-labeled with I-125-cholera toxin (segregating in caveolae) were is olated as described previously. Following incubation of these labeled membr anes in the presence of nucleotide(s) and cytosol, a significant proportion of the initially membrane-associated radioactivity was released into the i ncubation medium in sedimentable form (14x10(6) g), Results of biochemical, morphological, and fractionation analysis of the material containing the r eleased radioactivity directly demonstrated that caveolae are plasma membra ne domains involved in an endocytic process and resulting in the formation of caveolae-derived vesicles. In addition, these studies allowed a direct comparison of caveolae- and cla thrin-coated pit-mediated endocytosis and reveal that these two processes d iverge in terms of kinetics, cytosol and nucleotide requirements as well as in terms of the density and size of the endocytic vesicles formed.