Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Oct 1;123(1):67–77. doi: 10.1083/jcb.123.1.67

Assembly and targeting of adaptin chimeras in transfected cells

PMCID: PMC2119806  PMID: 8408206

Abstract

Adaptors are the components of clathrincoated pits and vesicles that attach the clathrin to the membrane. There are two types of adaptors in the cell: one associated with the plasma membrane and one associated with the TGN. Both adaptors are heterotetramers consisting of two adaptins (alpha and beta for the plasma membrane; gamma and beta' for the TGN), plus two smaller proteins. The COOH-terminal domains of the adaptins form appendages that resemble ears, connected by flexible hinges. Unlike the other adaptor components, the COOH termini of the alpha- and gamma-adaptins show no homology with each other, suggesting that they might provide the signal that directs the adaptors to the appropriate membrane. To test this possibility, the COOH-terminal ears were switched between alpha- and gamma-adaptins and were also deleted. All of the constructs contained the bovine gamma-adaptin hinge, enabling them to be detected with a species-specific antibody against this region when transfected into rat fibroblasts. Immunoprecipitation indicated that the engineered adaptins were still fully capable of assembling into adaptor complexes. Immunofluorescence revealed that in spite of their modified ears, the constructs were still able to be recruited onto the appropriate membrane; however, the ear-minus constructs gave increased cytoplasmic staining, and replacing the gamma- adaptin ear with the alpha-adaptin ear caused a small amount of colocalization with endogenous alpha-adaptin in some cells. Thus, the major targeting determinant appears to reside in the adaptor "head," while the ears may stabilize the association of adaptors with the membrane.

Full Text

The Full Text of this article is available as a PDF (3.9 MB).

Selected References

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

  1. Ahle S., Mann A., Eichelsbacher U., Ungewickell E. Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane. EMBO J. 1988 Apr;7(4):919–929. doi: 10.1002/j.1460-2075.1988.tb02897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahle S., Ungewickell E. Identification of a clathrin binding subunit in the HA2 adaptor protein complex. J Biol Chem. 1989 Nov 25;264(33):20089–20093. [PubMed] [Google Scholar]
  3. Beck K. A., Keen J. H. Self-association of the plasma membrane-associated clathrin assembly protein AP-2. J Biol Chem. 1991 Mar 5;266(7):4437–4441. [PubMed] [Google Scholar]
  4. Beltzer J. P., Spiess M. In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles. EMBO J. 1991 Dec;10(12):3735–3742. doi: 10.1002/j.1460-2075.1991.tb04942.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Donaldson J. G., Kahn R. A., Lippincott-Schwartz J., Klausner R. D. Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein. Science. 1991 Nov 22;254(5035):1197–1199. doi: 10.1126/science.1957170. [DOI] [PubMed] [Google Scholar]
  6. Glickman J. N., Conibear E., Pearse B. M. Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor. EMBO J. 1989 Apr;8(4):1041–1047. doi: 10.1002/j.1460-2075.1989.tb03471.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Heuser J. E., Keen J. Deep-etch visualization of proteins involved in clathrin assembly. J Cell Biol. 1988 Sep;107(3):877–886. doi: 10.1083/jcb.107.3.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Keen J. H., Beck K. A. Identification of the clathrin-binding domain of assembly protein AP-2. Biochem Biophys Res Commun. 1989 Jan 16;158(1):17–23. doi: 10.1016/s0006-291x(89)80170-6. [DOI] [PubMed] [Google Scholar]
  9. Kirchhausen T., Davis A. C., Frucht S., Greco B. O., Payne G. S., Tubb B. AP17 and AP19, the mammalian small chains of the clathrin-associated protein complexes show homology to Yap17p, their putative homolog in yeast. J Biol Chem. 1991 Jun 15;266(17):11153–11157. [PubMed] [Google Scholar]
  10. Kirchhausen T., Nathanson K. L., Matsui W., Vaisberg A., Chow E. P., Burne C., Keen J. H., Davis A. E. Structural and functional division into two domains of the large (100- to 115-kDa) chains of the clathrin-associated protein complex AP-2. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2612–2616. doi: 10.1073/pnas.86.8.2612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lobel P., Fujimoto K., Ye R. D., Griffiths G., Kornfeld S. Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis. Cell. 1989 Jun 2;57(5):787–796. doi: 10.1016/0092-8674(89)90793-9. [DOI] [PubMed] [Google Scholar]
  12. Luzio J. P., Brake B., Banting G., Howell K. E., Braghetta P., Stanley K. K. Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38). Biochem J. 1990 Aug 15;270(1):97–102. doi: 10.1042/bj2700097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mahaffey D. T., Peeler J. S., Brodsky F. M., Anderson R. G. Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity. J Biol Chem. 1990 Sep 25;265(27):16514–16520. [PubMed] [Google Scholar]
  14. Matsui W., Kirchhausen T. Stabilization of clathrin coats by the core of the clathrin-associated protein complex AP-2. Biochemistry. 1990 Dec 4;29(48):10791–10798. doi: 10.1021/bi00500a011. [DOI] [PubMed] [Google Scholar]
  15. Nakayama Y., Goebl M., O'Brine Greco B., Lemmon S., Pingchang Chow E., Kirchhausen T. The medium chains of the mammalian clathrin-associated proteins have a homolog in yeast. Eur J Biochem. 1991 Dec 5;202(2):569–574. doi: 10.1111/j.1432-1033.1991.tb16409.x. [DOI] [PubMed] [Google Scholar]
  16. Pearse B. M. Receptors compete for adaptors found in plasma membrane coated pits. EMBO J. 1988 Nov;7(11):3331–3336. doi: 10.1002/j.1460-2075.1988.tb03204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  18. Peeler J. S., Donzell W. C., Anderson R. G. The appendage domain of the AP-2 subunit is not required for assembly or invagination of clathrin-coated pits. J Cell Biol. 1993 Jan;120(1):47–54. doi: 10.1083/jcb.120.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ponnambalam S., Robinson M. S., Jackson A. P., Peiperl L., Parham P. Conservation and diversity in families of coated vesicle adaptins. J Biol Chem. 1990 Mar 25;265(9):4814–4820. [PubMed] [Google Scholar]
  20. Robinson M. S. 100-kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies. J Cell Biol. 1987 Apr;104(4):887–895. doi: 10.1083/jcb.104.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Robinson M. S. Adaptins. Trends Cell Biol. 1992 Oct;2(10):293–297. doi: 10.1016/0962-8924(92)90118-7. [DOI] [PubMed] [Google Scholar]
  22. Robinson M. S. Cloning and expression of gamma-adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus. J Cell Biol. 1990 Dec;111(6 Pt 1):2319–2326. doi: 10.1083/jcb.111.6.2319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Robinson M. S. Cloning of cDNAs encoding two related 100-kD coated vesicle proteins (alpha-adaptins). J Cell Biol. 1989 Mar;108(3):833–842. doi: 10.1083/jcb.108.3.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Robinson M. S., Kreis T. E. Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators. Cell. 1992 Apr 3;69(1):129–138. doi: 10.1016/0092-8674(92)90124-u. [DOI] [PubMed] [Google Scholar]
  25. Schröder S., Ungewickell E. Subunit interaction and function of clathrin-coated vesicle adaptors from the Golgi and the plasma membrane. J Biol Chem. 1991 Apr 25;266(12):7910–7918. [PubMed] [Google Scholar]
  26. Trowbridge I. S. Endocytosis and signals for internalization. Curr Opin Cell Biol. 1991 Aug;3(4):634–641. doi: 10.1016/0955-0674(91)90034-v. [DOI] [PubMed] [Google Scholar]
  27. Wong D. H., Brodsky F. M. 100-kD proteins of Golgi- and trans-Golgi network-associated coated vesicles have related but distinct membrane binding properties. J Cell Biol. 1992 Jun;117(6):1171–1179. doi: 10.1083/jcb.117.6.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES