Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Jul 1;101(1):12–18. doi: 10.1083/jcb.101.1.12

Solubilization of proteins from bovine brain coated vesicles by protein perturbants and Triton X-100

PMCID: PMC2113649  PMID: 2861205

Abstract

To identify integral and peripheral membrane proteins, highly purified coated vesicles from bovine brain were exposed to solutions of various pH, ionic strength, and concentrations of the nonionic detergent Triton X-100. At pH 10.0 or above most major proteins were liberated, but four minor polypeptides sedimented with the vesicles. From quantitative analysis of phospholipids in the pellet and extract, we determined that at a pH of up to 12 all phospholipids could be recovered in the pellet. Electron microscopic examination of coated vesicles at pH 12.0 showed all vesicles devoid of coat structures. Treatment with high ionic strength solutions (0-1.0 M KCl) at pH 6.5-8.5 also liberated all major proteins, except tubulin, which remained sedimentable. The addition of Triton X-100 to coated vesicles or to stripped vesicles from which 90% of the clathrin had been removed resulted in the release of four distinct polypeptides of approximate Mr 38,000, 29,000, 24,000 and 10,000. The 38,000-D polypeptide (pK approximately 5.0), which represents approximately 50% of the protein liberated by Triton X-100, appears to be a glycoprotein on the basis of its reaction with periodic acid-Schiff reagent. Extraction of 90% of the clathrin followed by extraction of 90% of the phospholipids with Triton X-100 produced a protein residue that remained sedimentable and consisted of structures that appeared to be shrunken stripped vesicles. Together our data indicate that most of the major polypeptides of brain coated vesicles behave as peripheral membrane proteins and at least four polypeptides behave as integral membrane proteins. By use of a monoclonal antibody, we have identified one of these polypeptides (38,000 mol wt) as a marker for a subpopulation of calf brain coated vesicles.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

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

  1. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  2. Batteiger B., Newhall W. J., 5th, Jones R. B. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods. 1982 Dec 30;55(3):297–307. doi: 10.1016/0022-1759(82)90089-8. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Eschenbruch M., Bürk R. R. Experimentally improved reliability of ultrasensitive silver staining of protein in polyacrylamide gels. Anal Biochem. 1982 Sep 1;125(1):96–99. doi: 10.1016/0003-2697(82)90387-6. [DOI] [PubMed] [Google Scholar]
  5. Fenner C., Traut R. R., Mason D. T., Wikman-Coffelt J. Quantification of Coomassie Blue stained proteins in polyacrylamide gels based on analyses of eluted dye. Anal Biochem. 1975 Feb;63(2):595–602. doi: 10.1016/0003-2697(75)90386-3. [DOI] [PubMed] [Google Scholar]
  6. Hanspal M., Luna E., Branton D. The association of clathrin fragments with coated vesicle membranes. J Biol Chem. 1984 Sep 10;259(17):11075–11082. [PubMed] [Google Scholar]
  7. Kanaseki T., Kadota K. The "vesicle in a basket". A morphological study of the coated vesicle isolated from the nerve endings of the guinea pig brain, with special reference to the mechanism of membrane movements. J Cell Biol. 1969 Jul;42(1):202–220. doi: 10.1083/jcb.42.1.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kartenbeck J., Schwechheimer K., Moll R., Franke W. W. Attachment of vimentin filaments to desmosomal plaques in human meningiomal cells and arachnoidal tissue. J Cell Biol. 1984 Mar;98(3):1072–1081. doi: 10.1083/jcb.98.3.1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Kelly W. G., Passaniti A., Woods J. W., Daiss J. L., Roth T. F. Tubulin as a molecular component of coated vesicles. J Cell Biol. 1983 Oct;97(4):1191–1199. doi: 10.1083/jcb.97.4.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Kumar N., Blumenthal R., Henkart M., Weinstein J. N., Klausner R. D. Aggregation and calcium-induced fusion of phosphatidylcholine vesicle-tubulin complexes. J Biol Chem. 1982 Dec 25;257(24):15137–15144. [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [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. 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]
  16. 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]
  17. 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]
  18. Rubenstein J. L., Fine R. E., Luskey B. D., Rothman J. E. Purification of coated vesicles by agarose gel electrophoresis. J Cell Biol. 1981 May;89(2):357–361. doi: 10.1083/jcb.89.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Steck T. L., Yu J. Selective solubilization of proteins from red blood cell membranes by protein perturbants. J Supramol Struct. 1973;1(3):220–232. doi: 10.1002/jss.400010307. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. Wiedenmann B., Mimms L. T. Tubulin is a major protein constituent of bovine brain coated vesicles. Biochem Biophys Res Commun. 1983 Aug 30;115(1):303–311. doi: 10.1016/0006-291x(83)91004-5. [DOI] [PubMed] [Google Scholar]
  24. Woodward M. P., Roth T. F. Influence of buffer ions and divalent cations on coated vesicle disassembly and reassembly. J Supramol Struct. 1979;11(2):237–250. doi: 10.1002/jss.400110213. [DOI] [PubMed] [Google Scholar]

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

RESOURCES