Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Mar;84(5):1137–1141. doi: 10.1073/pnas.84.5.1137

Fusion between endocytic vesicles in a cell-free system.

W A Braell
PMCID: PMC304381  PMID: 3469657

Abstract

Cell-free fusion between endocytic vesicles has been obtained in a sensitive assay based on the avidin-biotin binding reaction. Chinese hamster ovary cells are allowed to endocytose either avidin-linked beta-galactosidase or biotinylated IgG. Postnuclear supernatant extracts prepared from these cells are incubated at 37 degrees C in the presence of an ATP-regenerating system. Fusion between vesicles from the two extracts permits the avidin-biotin association to occur, so that the amount of avidin-enzyme-biotinylated IgG complex produced is proportional to the amount of fusion between vesicles. The amount of complex formed is measured, after detergent lysis of the vesicles, by an ELISA technique, using a fluorogenic substrate for beta-galactosidase. The fusion process requires ATP hydrolysis and specific cytosolic proteins. The vesicles that fuse appear to be endocytic vesicles populated by the endocytosed proteins within 5 min of their internalization at 37 degrees C. Ionophores and weak bases do not inhibit fusion, suggesting that a pH gradient across the vesicle membrane is not crucial for the fusion process.

Full text

PDF
1137

Images in this article

1137Image
on p.1137

Selected References

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

  1. Adams C. J., Maurey K. M., Storrie B. Exocytosis of pinocytic contents by Chinese hamster ovary cells. J Cell Biol. 1982 Jun;93(3):632–637. doi: 10.1083/jcb.93.3.632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altstiel L., Branton D. Fusion of coated vesicles with lysosomes: measurement with a fluorescence assay. Cell. 1983 Mar;32(3):921–929. doi: 10.1016/0092-8674(83)90077-6. [DOI] [PubMed] [Google Scholar]
  3. Balch W. E., Dunphy W. G., Braell W. A., Rothman J. E. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell. 1984 Dec;39(2 Pt 1):405–416. doi: 10.1016/0092-8674(84)90019-9. [DOI] [PubMed] [Google Scholar]
  4. Besterman J. M., Low R. B. Endocytosis: a review of mechanisms and plasma membrane dynamics. Biochem J. 1983 Jan 15;210(1):1–13. doi: 10.1042/bj2100001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Brown M. S., Anderson R. G., Goldstein J. L. Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell. 1983 Mar;32(3):663–667. doi: 10.1016/0092-8674(83)90052-1. [DOI] [PubMed] [Google Scholar]
  7. Davey J., Hurtley S. M., Warren G. Reconstitution of an endocytic fusion event in a cell-free system. Cell. 1985 Dec;43(3 Pt 2):643–652. doi: 10.1016/0092-8674(85)90236-3. [DOI] [PubMed] [Google Scholar]
  8. Dunn W. A., Hubbard A. L., Aronson N. N., Jr Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of 125I-asialofetuin by the perfused rat liver. J Biol Chem. 1980 Jun 25;255(12):5971–5978. [PubMed] [Google Scholar]
  9. Forgac M., Cantley L., Wiedenmann B., Altstiel L., Branton D. Clathrin-coated vesicles contain an ATP-dependent proton pump. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1300–1303. doi: 10.1073/pnas.80.5.1300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Galloway C. J., Dean G. E., Marsh M., Rudnick G., Mellman I. Acidification of macrophage and fibroblast endocytic vesicles in vitro. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3334–3338. doi: 10.1073/pnas.80.11.3334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Green N. M. Avidin. Adv Protein Chem. 1975;29:85–133. doi: 10.1016/s0065-3233(08)60411-8. [DOI] [PubMed] [Google Scholar]
  12. Green N. M., Toms E. J. The properties of subunits of avidin coupled to sepharose. Biochem J. 1973 Aug;133(4):687–700. doi: 10.1042/bj1330687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heitz J. R., Anderson C. D., Anderson B. M. Inactivation of yeast alcohol dehydrogenase by N-alkylmaleimides. Arch Biochem Biophys. 1968 Sep 20;127(1):627–636. doi: 10.1016/0003-9861(68)90271-3. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Marsh M., Bolzau E., Helenius A. Penetration of Semliki Forest virus from acidic prelysosomal vacuoles. Cell. 1983 Mar;32(3):931–940. doi: 10.1016/0092-8674(83)90078-8. [DOI] [PubMed] [Google Scholar]
  16. Maxfield F. R. Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts. J Cell Biol. 1982 Nov;95(2 Pt 1):676–681. doi: 10.1083/jcb.95.2.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. O'Sullivan M. J., Gnemmi E., Morris D., Chieregatti G., Simmonds A. D., Simmons M., Bridges J. W., Marks V. Comparison of two methods of preparing enzyme-antibody conjugates: application of these conjugates for enzyme immunoassay. Anal Biochem. 1979 Nov 15;100(1):100–108. doi: 10.1016/0003-2697(79)90117-9. [DOI] [PubMed] [Google Scholar]
  18. Poole B., Ohkuma S. Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages. J Cell Biol. 1981 Sep;90(3):665–669. doi: 10.1083/jcb.90.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rothman J. E., Schmid S. L. Enzymatic recycling of clathrin from coated vesicles. Cell. 1986 Jul 4;46(1):5–9. doi: 10.1016/0092-8674(86)90852-4. [DOI] [PubMed] [Google Scholar]
  20. Schmid S. L., Braell W. A., Schlossman D. M., Rothman J. E. A role for clathrin light chains in the recognition of clathrin cages by 'uncoating ATPase'. Nature. 1984 Sep 20;311(5983):228–231. doi: 10.1038/311228a0. [DOI] [PubMed] [Google Scholar]
  21. Steinman R. M., Mellman I. S., Muller W. A., Cohn Z. A. Endocytosis and the recycling of plasma membrane. J Cell Biol. 1983 Jan;96(1):1–27. doi: 10.1083/jcb.96.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stone D. K., Xie X. S., Racker E. An ATP-driven proton pump in clathrin-coated vesicles. J Biol Chem. 1983 Apr 10;258(7):4059–4062. [PubMed] [Google Scholar]
  23. Storrie B., Pool R. R., Jr, Sachdeva M., Maurey K. M., Oliver C. Evidence for both prelysosomal and lysosomal intermediates in endocytic pathways. J Cell Biol. 1984 Jan;98(1):108–115. doi: 10.1083/jcb.98.1.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES