Purification of clathrin heavy and light chain from Dictyostelium 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 approximately 2 mg of clathrin triskelions from 150-200 g of Dictyostelium cells. One additional step purified the 30-kDa clathrin light chain to homogeneity. Glycerol gradient centrifugation was used to determine an S value of 7.9 for purified clathrin. Rotary shadowed images of Dictyostelium clathrin 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 in Dictyostelium clathrin demonstrates the potential of this model organism for the study of clathrin structure and function.
Riddelle-Spencer
K S
KS
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
O'Halloran
T J
TJ
D000818:Animals; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002966:Clathrin / Q000737:chemistry* / Q000302:isolation & purification* / Q000648:ultrastructure; D033922:Clathrin Heavy Chains; D004023:Dictyostelium / Q000737:chemistry*; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008970:Molecular Weight
D002966:Clathrin; D046911:Macromolecular Substances; D033922:Clathrin Heavy Chains; D017886:Durapatite