Isolation and Preliminary Characterization of Clathrin-Associated Proteins

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Abstract
Clathrin-associated proteins were separated from clathrin under various clathrin-denaturing conditions, i.e., heating, freezing and isoelectric precipitation. The proteins retained biological activity; they were purified further by affinity chromatography on calmodulin-conjugated CNBr-Sepharose 4B and used for antibody purification. The affinity-purified anti-(clathrin-associated proteins) antibodies gave a fluorescent-dotted pattern in cultured [human] fibroblasts consistent with the known distribution of clathrin. Chemical cross-linking of pure clathrin-associated proteins indicated that these polypeptides existed as monomers in solution, each possessing Ca2+-dependent affinity for calmodulin to which they bind in a 1:1 molar ratio. Chymotryptic treatment of coated vesicles selectively cleaved the clathrin-associated proteins into a 15,000-18,000-MW doublet polypetide. These subfragments retained their Ca2+-dependent affinity for calmodulin. Clathrin-associated proteins probably have a regulatory role in clathrin assemblies.