Purification of clathrin heavy and light chain from Dictyostelium discoideum

Protein Expression and Purification
Kathryn Riddelle-Spencer, Theresa J O'Halloran

Abstract

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.

References

Sep 1, 1979·Proceedings of the National Academy of Sciences of the United States of America·H TowbinJ Gordon
May 1, 1992·DNA and Cell Biology·T J O'Halloran, R G Anderson
Jul 1, 1985·Journal of Ultrastructure Research·J Heuser, T Kirchhausen
Dec 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·T KirchhausenJ Brosius
May 1, 1981·Journal of Ultrastructure Research·J A SwansonJ T Bonner
May 1, 1983·Proceedings of the National Academy of Sciences of the United States of America·T KirchhausenF M Brodsky
Jan 1, 1984·The Journal of Cell Biology·S C Mueller, D Branton
Nov 1, 1984·The Journal of Cell Biology·T Kirchhausen, S C Harrison
Mar 1, 1983·The Journal of Cell Biology·F M BrodskyP Parham
Mar 1, 1981·Cell·T Kirchhausen, S C Harrison
Jan 29, 1981·Nature·E Ungewickell, D Branton
Aug 1, 1994·Current Opinion in Cell Biology·M S Robinson
Jan 1, 1993·Critical Reviews in Biochemistry and Molecular Biology·U Pley, P Parham
Aug 5, 1997·Proceedings of the National Academy of Sciences of the United States of America·M L Niswonger, T J O'Halloran

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