Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1986 Dec 1;5(12):3143–3149. doi: 10.1002/j.1460-2075.1986.tb04621.x

Purification and properties of a new clathrin assembly protein.

S Ahle, E Ungewickell
PMCID: PMC1167304  PMID: 3816757

Abstract

A clathrin assembly protein (AP180) has been purified and characterized from coated vesicles of bovine brain. This protein has hitherto escaped detection because in SDS-gel electrophoresis it is obscured by the 180 kd heavy chain of clathrin. Despite the similarity in electrophoretic mobility, AP180 differs from clathrin in both its subunit and native mol. wt, as well as hydrodynamic properties, surface charge and tryptic peptide composition. It also appears immunologically distinct from clathrin, since neither a polyclonal antiserum nor a monoclonal antibody, that have been shown to be specific for AP180, cross-react with the heavy chain of clathrin. AP180 binds to clathrin triskelia and thereby promotes clathrin assembly into regular polyhedral structures of narrow size-distribution (60-90 nm), reminiscent of the surface coat of coated vesicles. In this respect AP180 bears a functional resemblance to the 100-110 kd clathrin assembly polypeptides that have been previously described.

Full text

PDF
3143

Images in this article

3144Fig. 2.
on p.3144
3144Fig. 3.
on p.3144
3144Fig. 4.
on p.3144
3145Fig. 5.
on p.3145
3146Fig. 8.
on p.3146
3146Fig. 9.
on p.3146

Selected References

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

  1. Bernardi G., Kawasaki T. Chromatography of polypeptides and proteins on hydroxyapatite columns. Biochim Biophys Acta. 1968 Aug 13;160(3):301–310. doi: 10.1016/0005-2795(68)90203-1. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Bretscher M. S. Major human erythrocyte glycoprotein spans the cell membrane. Nat New Biol. 1971 Jun 23;231(25):229–232. doi: 10.1038/newbio231229a0. [DOI] [PubMed] [Google Scholar]
  4. Campbell C., Squicciarini J., Shia M., Pilch P. F., Fine R. E. Identification of a protein kinase as an intrinsic component of rat liver coated vesicles. Biochemistry. 1984 Sep 11;23(19):4420–4426. doi: 10.1021/bi00314a028. [DOI] [PubMed] [Google Scholar]
  5. Clezardin P., McGregor J. L., Manach M., Boukerche H., Dechavanne M. One-step procedure for the rapid isolation of mouse monoclonal antibodies and their antigen binding fragments by fast protein liquid chromatography on a mono Q anion-exchange column. J Chromatogr. 1985 Jan 25;319(1):67–77. doi: 10.1016/s0021-9673(01)90540-0. [DOI] [PubMed] [Google Scholar]
  6. Debus E., Weber K., Osborn M. Monoclonal antibodies to desmin, the muscle-specific intermediate filament protein. EMBO J. 1983;2(12):2305–2312. doi: 10.1002/j.1460-2075.1983.tb01738.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Elder J. H., Pickett R. A., 2nd, Hampton J., Lerner R. A. Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein. J Biol Chem. 1977 Sep 25;252(18):6510–6515. [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. Goldstein J. L., Brown M. S., Anderson R. G., Russell D. W., Schneider W. J. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1985;1:1–39. doi: 10.1146/annurev.cb.01.110185.000245. [DOI] [PubMed] [Google Scholar]
  10. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  11. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  12. Keen J. H., Black M. M. The phosphorylation of coated membrane proteins in intact neurons. J Cell Biol. 1986 Apr;102(4):1325–1333. doi: 10.1083/jcb.102.4.1325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Keen J. H., Willingham M. C., Pastan I. H. Clathrin-coated vesicles: isolation, dissociation and factor-dependent reassociation of clathrin baskets. Cell. 1979 Feb;16(2):303–312. doi: 10.1016/0092-8674(79)90007-2. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Lehrman M. A., Goldstein J. L., Brown M. S., Russell D. W., Schneider W. J. Internalization-defective LDL receptors produced by genes with nonsense and frameshift mutations that truncate the cytoplasmic domain. Cell. 1985 Jul;41(3):735–743. doi: 10.1016/s0092-8674(85)80054-4. [DOI] [PubMed] [Google Scholar]
  17. Mann K. G., Fish W. W. Protein polypeptide chain molecular weights by gel chromatography in guanidinium chloride. Methods Enzymol. 1972;26:28–42. doi: 10.1016/s0076-6879(72)26004-9. [DOI] [PubMed] [Google Scholar]
  18. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  19. Pauloin A., Jollès P. Internal control of the coated vesicle pp50-specific kinase complex. Nature. 1984 Sep 20;311(5983):265–267. doi: 10.1038/311265a0. [DOI] [PubMed] [Google Scholar]
  20. Pearse B. M. Assembly of the mannose-6-phosphate receptor into reconstituted clathrin coats. EMBO J. 1985 Oct;4(10):2457–2460. doi: 10.1002/j.1460-2075.1985.tb03956.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pearse B. M., Bretscher M. S. Membrane recycling by coated vesicles. Annu Rev Biochem. 1981;50:85–101. doi: 10.1146/annurev.bi.50.070181.000505. [DOI] [PubMed] [Google Scholar]
  22. Pearse B. M., Robinson M. S. Purification and properties of 100-kd proteins from coated vesicles and their reconstitution with clathrin. EMBO J. 1984 Sep;3(9):1951–1957. doi: 10.1002/j.1460-2075.1984.tb02075.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Robinson M. S., Pearse B. M. Immunofluorescent localization of 100K coated vesicle proteins. J Cell Biol. 1986 Jan;102(1):48–54. doi: 10.1083/jcb.102.1.48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Ungewickell E. Biochemical and immunological studies on clathrin light chains and their binding sites on clathrin triskelions. EMBO J. 1983;2(8):1401–1408. doi: 10.1002/j.1460-2075.1983.tb01598.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Ungewickell E. Characterization of clathrin and clathrin-associated proteins. Biochem Soc Trans. 1984 Dec;12(6):978–980. doi: 10.1042/bst0120978. [DOI] [PubMed] [Google Scholar]
  29. Ungewickell E. The 70-kd mammalian heat shock proteins are structurally and functionally related to the uncoating protein that releases clathrin triskelia from coated vesicles. EMBO J. 1985 Dec 16;4(13A):3385–3391. doi: 10.1002/j.1460-2075.1985.tb04094.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Valentine R. C., Shapiro B. M., Stadtman E. R. Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. Biochemistry. 1968 Jun;7(6):2143–2152. doi: 10.1021/bi00846a017. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Williams J. G., Gratzer W. B. Limitations of the detergent-polyacrylamide gel electrophoresis method for molecular weight determination of proteins. J Chromatogr. 1971 Apr 22;57(1):121–125. doi: 10.1016/0021-9673(71)80013-4. [DOI] [PubMed] [Google Scholar]
  33. Woodward M. P., Roth T. F. Coated vesicles: characterization, selective dissociation, and reassembly. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4394–4398. doi: 10.1073/pnas.75.9.4394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zaremba S., Keen J. H. Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats. J Cell Biol. 1983 Nov;97(5 Pt 1):1339–1347. doi: 10.1083/jcb.97.5.1339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zaremba S., Keen J. H. Limited proteolytic digestion of coated vesicle assembly polypeptides abolishes reassembly activity. J Cell Biochem. 1985;28(1):47–58. doi: 10.1002/jcb.240280108. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES