Biochemical and immunological studies on clathrin light chains and their binding sites on clathrin triskelions (original) (raw)

Abstract

Clathrin light chains from bovine brain tissue (LC alpha and LC beta) are monomeric proteins with an average mol. wt. of approximately 33,000, as determined by sedimentation equilibrium. Solution studies on purified light chains indicate a large Stokes radius (Re = 3.3 nm) and little defined secondary structure. Both light chains bind specifically and with high affinity (KA approximately 5 x 10(7)/M) to overlapping sites on clathrin heavy chains. These binding sites are contained within a 125,000 dalton heavy chain fragment that forms truncated triskelions with legs, 15 nm shorter than those of intact triskelions. As judged by immuno-electron microscopy, light chain-specific IgG molecules bind mostly to the center of triskelions, but there are also sites that are scattered some 16 nm along the proximal part of triskelion legs. From heterologous binding experiments using human placenta light chains and heavy chain fragments from bovine brain clathrin, it is concluded that the domains of light and heavy chains that are involved in the interaction are conserved across tissue and species boundaries.

1401

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ackers G. K. Analytical gel chromatography of proteins. Adv Protein Chem. 1970;24:343–446. doi: 10.1016/s0065-3233(08)60245-4. [DOI] [PubMed] [Google Scholar]
  2. Bailey K., Gutfreund H., Ogston A. G. Molecular weight of tropomyosin from rabbit muscle. Biochem J. 1948;43(2):279–281. doi: 10.1042/bj0430279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bretscher M. S., Thomson J. N., Pearse B. M. Coated pits act as molecular filters. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4156–4159. doi: 10.1073/pnas.77.7.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brodsky F. M., Holmes N. J., Parham P. Tropomyosin-like properties of clathrin light chains allow a rapid, high-yield purification. J Cell Biol. 1983 Mar;96(3):911–914. doi: 10.1083/jcb.96.3.911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cheung W. Y. Calmodulin plays a pivotal role in cellular regulation. Science. 1980 Jan 4;207(4426):19–27. doi: 10.1126/science.6243188. [DOI] [PubMed] [Google Scholar]
  6. Crowther R. A., Pearse B. M. Assembly and packing of clathrin into coats. J Cell Biol. 1981 Dec;91(3 Pt 1):790–797. doi: 10.1083/jcb.91.3.790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fisher P. A., Berrios M., Blobel G. Isolation and characterization of a proteinaceous subnuclear fraction composed of nuclear matrix, peripheral lamina, and nuclear pore complexes from embryos of Drosophila melanogaster. J Cell Biol. 1982 Mar;92(3):674–686. doi: 10.1083/jcb.92.3.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  9. Greenfield N., Fasman G. D. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry. 1969 Oct;8(10):4108–4116. doi: 10.1021/bi00838a031. [DOI] [PubMed] [Google Scholar]
  10. Herzog W., Weber K. Fractionation of brain microtubule-associated proteins. Isolation of two different proteins which stimulate tubulin polymerization in vitro. Eur J Biochem. 1978 Dec 1;92(1):1–8. doi: 10.1111/j.1432-1033.1978.tb12716.x. [DOI] [PubMed] [Google Scholar]
  11. Kartenbeck J., Schmid E., Müller H., Franke W. W. Immunological identification and localization of clathrin and coated vesicles in cultured cells and in tissues. Exp Cell Res. 1981 May;133(1):191–211. doi: 10.1016/0014-4827(81)90369-4. [DOI] [PubMed] [Google Scholar]
  12. Kawahara K., Tanford C. Viscosity and density of aqueous solutions of urea and guanidine hydrochloride. J Biol Chem. 1966 Jul 10;241(13):3228–3232. [PubMed] [Google Scholar]
  13. Keen J. H., Willingham M. C., Pastan I. Clathrin and coated vesicle proteins Immunological characterization. J Biol Chem. 1981 Mar 10;256(5):2538–2544. [PubMed] [Google Scholar]
  14. Kirchhausen T., Harrison S. C., Parham P., Brodsky F. M. Location and distribution of the light chains in clathrin trimers. Proc Natl Acad Sci U S A. 1983 May;80(9):2481–2485. doi: 10.1073/pnas.80.9.2481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kirchhausen T., Harrison S. C. Protein organization in clathrin trimers. Cell. 1981 Mar;23(3):755–761. doi: 10.1016/0092-8674(81)90439-6. [DOI] [PubMed] [Google Scholar]
  16. Kuntz I. D., Jr, Kauzmann W. Hydration of proteins and polypeptides. Adv Protein Chem. 1974;28:239–345. doi: 10.1016/s0065-3233(08)60232-6. [DOI] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Laskey R. A., Mills A. D. Enhanced autoradiographic detection of 32P and 125I using intensifying screens and hypersensitized film. FEBS Lett. 1977 Oct 15;82(2):314–316. doi: 10.1016/0014-5793(77)80609-1. [DOI] [PubMed] [Google Scholar]
  19. Lisanti M. P., Shapiro L. S., Moskowitz N., Hua E. L., Puszkin S., Schook W. Isolation and preliminary characterization of clathrin-associated proteins. Eur J Biochem. 1982 Jul;125(2):463–470. doi: 10.1111/j.1432-1033.1982.tb06706.x. [DOI] [PubMed] [Google Scholar]
  20. Mendel-Hartvig I. B. A simple and rapid method for the isolation of peptides from sodium dodecyl sulfate-containing polyacrylamide gels. Anal Biochem. 1982 Mar 15;121(1):215–217. doi: 10.1016/0003-2697(82)90579-6. [DOI] [PubMed] [Google Scholar]
  21. Pearse B. M. Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1255–1259. doi: 10.1073/pnas.73.4.1255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pearse B. M. Coated vesicles from human placenta carry ferritin, transferrin, and immunoglobulin G. Proc Natl Acad Sci U S A. 1982 Jan;79(2):451–455. doi: 10.1073/pnas.79.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pearse B. M. Coated vesicles from pig brain: purification and biochemical characterization. J Mol Biol. 1975 Sep 5;97(1):93–98. doi: 10.1016/s0022-2836(75)80024-6. [DOI] [PubMed] [Google Scholar]
  24. Pearse B. M. On the structural and functional components of coated vesicles. J Mol Biol. 1978 Dec 25;126(4):803–812. doi: 10.1016/0022-2836(78)90021-9. [DOI] [PubMed] [Google Scholar]
  25. Pont M. J., Woods E. F. Denaturation of tropomyosin by guanidine hydrochloride. Int J Protein Res. 1971;3(4):177–183. doi: 10.1111/j.1399-3011.1971.tb01710.x. [DOI] [PubMed] [Google Scholar]
  26. Schmid S. L., Matsumoto A. K., Rothman J. E. A domain of clathrin that forms coats. Proc Natl Acad Sci U S A. 1982 Jan;79(1):91–95. doi: 10.1073/pnas.79.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stone D., Smillie L. B. The amino acid sequence of rabbit skeletal alpha-tropomyosin. The NH2-terminal half and complete sequence. J Biol Chem. 1978 Feb 25;253(4):1137–1148. [PubMed] [Google Scholar]
  28. Tack B. F., Dean J., Eilat D., Lorenz P. E., Schechter A. N. Tritium labeling of proteins to high specific radioactivity by reduction methylation. J Biol Chem. 1980 Sep 25;255(18):8842–8847. [PubMed] [Google Scholar]
  29. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tyler J. M., Branton D. Rotary shadowing of extended molecules dried from glycerol. J Ultrastruct Res. 1980 May;71(2):95–102. doi: 10.1016/s0022-5320(80)90098-2. [DOI] [PubMed] [Google Scholar]
  31. Unanue E. R., Ungewickell E., Branton D. The binding of clathrin triskelions to membranes from coated vesicles. Cell. 1981 Nov;26(3 Pt 1):439–446. doi: 10.1016/0092-8674(81)90213-0. [DOI] [PubMed] [Google Scholar]
  32. Ungewickell E., Branton D. Assembly units of clathrin coats. Nature. 1981 Jan 29;289(5796):420–422. doi: 10.1038/289420a0. [DOI] [PubMed] [Google Scholar]
  33. Ungewickell E., Unanue E. R., Branton D. Functional and structural studies on clathrin triskelions and baskets. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):723–731. doi: 10.1101/sqb.1982.046.01.069. [DOI] [PubMed] [Google Scholar]
  34. WADDELL W. J. A simple ultraviolet spectrophotometric method for the determination of protein. J Lab Clin Med. 1956 Aug;48(2):311–314. [PubMed] [Google Scholar]
  35. YPHANTIS D. A. EQUILIBRIUM ULTRACENTRIFUGATION OF DILUTE SOLUTIONS. Biochemistry. 1964 Mar;3:297–317. doi: 10.1021/bi00891a003. [DOI] [PubMed] [Google Scholar]