Purification of Clathrin Heavy and Light Chain fromDictyostelium discoideum

Clathrin, a protein important for endocytosis, is a hexamer composed of three heavy chains and three light chains. We report here the purification scheme used to isolate the clathrin protein from the simple eukaryote,Dictyostelium discoideum.Using a combination of differential centrifugation and column chromatography, we isolated ∼2 mg of clathrin triskelions from 150–200 g ofDictyosteliumcells. One additional step purified the 30-kDa clathrin light chain to homogeneity. Glycerol gradient centrifugation was used to determine anSvalue of 7.9 for purified clathrin. Rotary shadowed images ofDictyosteliumclathrin revealed trimeric molecules with extended legs measuring 48 ± 5 nm, similar in length to the legs of mammalian and yeast clathrin triskelions. The single clathrin light chain proved resistant to heat treatment, a property also similar to light chains from other species. The conservation of these physical properties inDictyosteliumclathrin demonstrates the potential of this model organism for the study of clathrin structure and function.