pmc.ncbi.nlm.nih.gov

Auxilin, a newly identified clathrin-associated protein in coated vesicles from bovine brain

Abstract

We have identified a new coat protein in clathrin-coated vesicles from bovine brain by urea-SDS gel electrophoresis. The protein was purified from Tris-solubilized coat proteins either by combination of hydroxyapatite chromatography and gel filtration or more rapidly in a single step by immunoaffinity chromatography. The purified protein binds to clathrin triskelia and thereby promotes clathrin assembly into regular 50-100-nm cages. We propose for the new protein the name auxilin (Latin auxilium, meaning support). Auxilin migrates as a 110-kD polypeptide in standard type SDS-PAGE, but in the presence of 6 M urea shifts to a position corresponding to 126 kD. Gel filtration in 6 M guanidinium hydrochloride gives a molecular weight of approximately 86,000. The native protein is monomeric in 0.5 M Tris. Antigenic reactivity and two-dimensional peptide maps gave no evidence of gross similarities between auxilin and any of the other known coated vesicle- associated proteins. Since the structural organization of auxilin does not resemble that of the ubiquitous heterotetrameric HA1 and HA2 adaptor complexes, that are believed to connect clathrin to receptors, it is unlikely that it functions as an adaptor. Immunoblotting did not reveal the presence of auxilin in tissues other than brain. If auxilin and AP 180 are indeed both confined to neuronal cells, as the immunochemical evidence suggests, it might be inferred that both serve to adapt clathrin-coated vesicles to an as yet undisclosed function unique to this cell type.

Full Text

The Full Text of this article is available as a PDF (3.4 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. 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]
  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. 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]
  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. 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]
  8. Irace G., Lippoldt R. E., Edelhoch H., Nandi P. K. Properties of clathrin coat structures. Biochemistry. 1982 Nov 9;21(23):5764–5769. doi: 10.1021/bi00266a006. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. Kirchhausen T., Scarmato P., Harrison S. C., Monroe J. J., Chow E. P., Mattaliano R. J., Ramachandran K. L., Smart J. E., Ahn A. H., Brosius J. Clathrin light chains LCA and LCB are similar, polymorphic, and share repeated heptad motifs. Science. 1987 Apr 17;236(4799):320–324. doi: 10.1126/science.3563513. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Morris S. A., Ahle S., Ungewickell E. Clathrin-coated vesicles. Curr Opin Cell Biol. 1989 Aug;1(4):684–690. doi: 10.1016/0955-0674(89)90034-3. [DOI] [PubMed] [Google Scholar]
  16. Morris S. A., Hannig K., Ungewickell E. Rapid purification of clathrin-coated vesicles by free-flow electrophoresis. Eur J Cell Biol. 1988 Dec;47(2):251–258. [PubMed] [Google Scholar]
  17. 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]
  18. Pearse B. M. Clathrin and coated vesicles. EMBO J. 1987 Sep;6(9):2507–2512. doi: 10.1002/j.1460-2075.1987.tb02536.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  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., Lippoldt R. E. Molecular characterization of the AP180 coated vesicle assembly protein. Biochemistry. 1988 Aug 9;27(16):6098–6104. doi: 10.1021/bi00416a040. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Schmid S. L., Rothman J. E. Enzymatic dissociation of clathrin cages in a two-stage process. J Biol Chem. 1985 Aug 25;260(18):10044–10049. [PubMed] [Google Scholar]
  27. Scopes R. K. Measurement of protein by spectrophotometry at 205 nm. Anal Biochem. 1974 May;59(1):277–282. doi: 10.1016/0003-2697(74)90034-7. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Ungewickell E., Oestergaard L. Identification of the clathrin assembly protein AP180 in crude calf brain extracts by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem. 1989 Jun;179(2):352–356. doi: 10.1016/0003-2697(89)90143-7. [DOI] [PubMed] [Google Scholar]
  32. 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]