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. 1988 Sep 1;107(3):877–886. doi: 10.1083/jcb.107.3.877

Deep-etch visualization of proteins involved in clathrin assembly

PMCID: PMC2115277  PMID: 3417785

Abstract

Assembly proteins were extracted from bovine brain clathrin-coated vesicles with 0.5 M Tris and purified by clathrin-Sepharose affinity chromatography, then adsorbed to mica and examined by freeze-etch electron microscopy. The fraction possessing maximal ability to promote clathrin polymerization, termed AP-2, was found to be a tripartite structure composed of a relatively large central mass flanked by two smaller mirror-symmetric appendages. Elastase treatment quantitatively removed the appendages and clipped 35 kD from the molecule's major approximately 105-kD polypeptides, indicating that the appendages are made from portions of these polypeptides. The remaining central masses no longer promote clathrin polymerization, suggesting that the appendages are somehow involved in the clathrin assembly reaction. The central masses are themselves relatively compact and brick-shaped, and are sufficiently large to contain two copies of the molecule's other major polypeptides (16- and 50-kD), as well as two copies of the approximately 70-kD protease-resistant portions of the major approximately 105-kD polypeptides. Thus the native molecule seems to be a dimeric, bilaterally symmetrical entity. Direct visualization of AP-2 binding to clathrin was accomplished by preparing mixtures of the two molecules in buffers that marginally inhibit AP-2 aggregation and cage assembly. This revealed numerous examples of AP-2 molecules binding to the so-called terminal domains of clathrin triskelions, consistent with earlier electron microscopic evidence that in fully assembled cages, the AP's attach centrally to inwardly-directed terminal domains of the clathrin molecule. This would place AP-2s between the clathrin coat and the enclosed membrane in whole coated vesicles. AP-2s linked to the membrane were also visualized by enzymatically removing the clathrin from brain coated vesicles, using purified 70 kD, uncoating ATPase plus ATP. This revealed several brick-shaped molecules attached to the vesicle membrane by short stalks. The exact stoichiometry of APs to clathrin in such vesicles, before and after uncoating, remains to be determined.

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Selected References

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  1. Ahle S., Ungewickell E. Purification and properties of a new clathrin assembly protein. EMBO J. 1986 Dec 1;5(12):3143–3149. doi: 10.1002/j.1460-2075.1986.tb04621.x. [DOI] [PMC free article] [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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Brown M. S., Goldstein J. L. A receptor-mediated pathway for cholesterol homeostasis. Science. 1986 Apr 4;232(4746):34–47. doi: 10.1126/science.3513311. [DOI] [PubMed] [Google Scholar]
  4. Crowther R. A., Finch J. T., Pearse B. M. On the structure of coated vesicles. J Mol Biol. 1976 Jun 5;103(4):785–798. doi: 10.1016/0022-2836(76)90209-6. [DOI] [PubMed] [Google Scholar]
  5. Fowler W. E., Erickson H. P. Trinodular structure of fibrinogen. Confirmation by both shadowing and negative stain electron microscopy. J Mol Biol. 1979 Oct 25;134(2):241–249. doi: 10.1016/0022-2836(79)90034-2. [DOI] [PubMed] [Google Scholar]
  6. Goodenough U., Heuser J. Structural comparison of purified dynein proteins with in situ dynein arms. J Mol Biol. 1984 Dec 25;180(4):1083–1118. doi: 10.1016/0022-2836(84)90272-9. [DOI] [PubMed] [Google Scholar]
  7. Heuser J. E. Procedure for freeze-drying molecules adsorbed to mica flakes. J Mol Biol. 1983 Sep 5;169(1):155–195. doi: 10.1016/s0022-2836(83)80179-x. [DOI] [PubMed] [Google Scholar]
  8. Heuser J., Kirchhausen T. Deep-etch views of clathrin assemblies. J Ultrastruct Res. 1985 Jul-Aug;92(1-2):1–27. doi: 10.1016/0889-1605(85)90123-5. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Keen J. H., Chestnut M. H., Beck K. A. The clathrin coat assembly polypeptide complex. Autophosphorylation and assembly activities. J Biol Chem. 1987 Mar 15;262(8):3864–3871. [PubMed] [Google Scholar]
  11. Keen J. H. Clathrin assembly proteins: affinity purification and a model for coat assembly. J Cell Biol. 1987 Nov;105(5):1989–1998. doi: 10.1083/jcb.105.5.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Kirchhausen T., Harrison S. C., Heuser J. Configuration of clathrin trimers: evidence from electron microscopy. J Ultrastruct Mol Struct Res. 1986 Mar;94(3):199–208. doi: 10.1016/0889-1605(86)90067-4. [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. Kirchhausen T., Harrison S. C. Structural domains of clathrin heavy chains. J Cell Biol. 1984 Nov;99(5):1725–1734. doi: 10.1083/jcb.99.5.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Norton P. A., Slayter H. S. Immune labeling of the D and E regions of human fibrinogen by electron microscopy. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1661–1665. doi: 10.1073/pnas.78.3.1661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Patzer E. J., Schlossman D. M., Rothman J. E. Release of clathrin from coated vesicles dependent upon a nucleoside triphosphate and a cytosol fraction. J Cell Biol. 1982 Apr;93(1):230–236. doi: 10.1083/jcb.93.1.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Pearse B. M., Crowther R. A. Structure and assembly of coated vesicles. Annu Rev Biophys Biophys Chem. 1987;16:49–68. doi: 10.1146/annurev.bb.16.060187.000405. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Prasad K., Yora T., Yano O., Lippoldt R. E., Edelhoch H., Saroff H. Purification and characterization of a molecular weight 100,000 coat protein from coated vesicles obtained from bovine brain. Biochemistry. 1986 Nov 4;25(22):6942–6947. doi: 10.1021/bi00370a030. [DOI] [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. Schlossman D. M., Schmid S. L., Braell W. A., Rothman J. E. An enzyme that removes clathrin coats: purification of an uncoating ATPase. J Cell Biol. 1984 Aug;99(2):723–733. doi: 10.1083/jcb.99.2.723. [DOI] [PMC free article] [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., Branton D. Assembly units of clathrin coats. Nature. 1981 Jan 29;289(5796):420–422. doi: 10.1038/289420a0. [DOI] [PubMed] [Google Scholar]
  27. Vigers G. P., Crowther R. A., Pearse B. M. Location of the 100 kd-50 kd accessory proteins in clathrin coats. EMBO J. 1986 Sep;5(9):2079–2085. doi: 10.1002/j.1460-2075.1986.tb04469.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vigers G. P., Crowther R. A., Pearse B. M. Three-dimensional structure of clathrin cages in ice. EMBO J. 1986 Mar;5(3):529–534. doi: 10.1002/j.1460-2075.1986.tb04242.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Virshup D. M., Bennett V. Clathrin-coated vesicle assembly polypeptides: physical properties and reconstitution studies with brain membranes. J Cell Biol. 1988 Jan;106(1):39–50. doi: 10.1083/jcb.106.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Weisel J. W., Stauffacher C. V., Bullitt E., Cohen C. A model for fibrinogen: domains and sequence. Science. 1985 Dec 20;230(4732):1388–1391. doi: 10.1126/science.4071058. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]

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