Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Mar 1;108(3):843–853. doi: 10.1083/jcb.108.3.843

Formation of coated vesicles from coated pits in broken A431 cells

PMCID: PMC2115393  PMID: 2564003

Abstract

Biochemical and morphological techniques were used to demonstrate the early steps in the endocytosis of transferrin in broken A431 cells. After binding 125I-transferrin, the cells were broken by scraping and then warmed. 125I-transferrin became inaccessible to exogenous anti- transferrin antibody providing a measure of the internalization process. Parallel morphological experiments using transferrin coupled to horseradish peroxidase confirmed internalization in broken cells. The process was characterized and compared with endocytosis in intact cells and showed many similar features. The system was used to show that both the appearance of new coated pits and the scission of coated pits to form coated vesicles were dependent on the addition of cytosol and ATP whereas invagination of pits was dependent on neither.

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

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

  1. Anderson R. G., Brown M. S., Beisiegel U., Goldstein J. L. Surface distribution and recycling of the low density lipoprotein receptor as visualized with antireceptor antibodies. J Cell Biol. 1982 Jun;93(3):523–531. doi: 10.1083/jcb.93.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Beckers C. J., Keller D. S., Balch W. E. Semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex. Cell. 1987 Aug 14;50(4):523–534. doi: 10.1016/0092-8674(87)90025-0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bretscher M. S., Thomson J. N. The morphology of endosomes in giant HeLa cells. Eur J Cell Biol. 1985 May;37:78–80. [PubMed] [Google Scholar]
  6. Carpenter G. Receptors for epidermal growth factor and other polypeptide mitogens. Annu Rev Biochem. 1987;56:881–914. doi: 10.1146/annurev.bi.56.070187.004313. [DOI] [PubMed] [Google Scholar]
  7. Ciechanover A., Schwartz A. L., Dautry-Varsat A., Lodish H. F. Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents. J Biol Chem. 1983 Aug 25;258(16):9681–9689. [PubMed] [Google Scholar]
  8. Dautry-Varsat A., Ciechanover A., Lodish H. F. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2258–2262. doi: 10.1073/pnas.80.8.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davis C. G., Lehrman M. A., Russell D. W., Anderson R. G., Brown M. S., Goldstein J. L. The J.D. mutation in familial hypercholesterolemia: amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors. Cell. 1986 Apr 11;45(1):15–24. doi: 10.1016/0092-8674(86)90533-7. [DOI] [PubMed] [Google Scholar]
  10. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  11. Geuze H. J., Slot J. W., Strous G. J., Peppard J., von Figura K., Hasilik A., Schwartz A. L. Intracellular receptor sorting during endocytosis: comparative immunoelectron microscopy of multiple receptors in rat liver. Cell. 1984 May;37(1):195–204. doi: 10.1016/0092-8674(84)90315-5. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Gundersen H. J. Stereology of arbitrary particles. A review of unbiased number and size estimators and the presentation of some new ones, in memory of William R. Thompson. J Microsc. 1986 Jul;143(Pt 1):3–45. [PubMed] [Google Scholar]
  14. Helenius A., Kartenbeck J., Simons K., Fries E. On the entry of Semliki forest virus into BHK-21 cells. J Cell Biol. 1980 Feb;84(2):404–420. doi: 10.1083/jcb.84.2.404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hesketh T. R., Moore J. P., Morris J. D., Taylor M. V., Rogers J., Smith G. A., Metcalfe J. C. A common sequence of calcium and pH signals in the mitogenic stimulation of eukaryotic cells. Nature. 1985 Feb 7;313(6002):481–484. doi: 10.1038/313481a0. [DOI] [PubMed] [Google Scholar]
  16. Heuser J. Three-dimensional visualization of coated vesicle formation in fibroblasts. J Cell Biol. 1980 Mar;84(3):560–583. doi: 10.1083/jcb.84.3.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hopkins C. R. The appearance and internalization of transferrin receptors at the margins of spreading human tumor cells. Cell. 1985 Jan;40(1):199–208. doi: 10.1016/0092-8674(85)90323-x. [DOI] [PubMed] [Google Scholar]
  18. Hopkins C. R., Trowbridge I. S. Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells. J Cell Biol. 1983 Aug;97(2):508–521. doi: 10.1083/jcb.97.2.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Klausner R. D., Ashwell G., van Renswoude J., Harford J. B., Bridges K. R. Binding of apotransferrin to K562 cells: explanation of the transferrin cycle. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2263–2266. doi: 10.1073/pnas.80.8.2263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kosaka T., Ikeda K. Reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of Drosophila melanogaster, shibirets1. J Cell Biol. 1983 Aug;97(2):499–507. doi: 10.1083/jcb.97.2.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lazarovits J., Roth M. A single amino acid change in the cytoplasmic domain allows the influenza virus hemagglutinin to be endocytosed through coated pits. Cell. 1988 Jun 3;53(5):743–752. doi: 10.1016/0092-8674(88)90092-x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Marsh M., Helenius A. Adsorptive endocytosis of Semliki Forest virus. J Mol Biol. 1980 Sep 25;142(3):439–454. doi: 10.1016/0022-2836(80)90281-8. [DOI] [PubMed] [Google Scholar]
  24. Moore M. S., Mahaffey D. T., Brodsky F. M., Anderson R. G. Assembly of clathrin-coated pits onto purified plasma membranes. Science. 1987 May 1;236(4801):558–563. doi: 10.1126/science.2883727. [DOI] [PubMed] [Google Scholar]
  25. Mostov K. E., Simister N. E. Transcytosis. Cell. 1985 Dec;43(2 Pt 1):389–390. doi: 10.1016/0092-8674(85)90166-7. [DOI] [PubMed] [Google Scholar]
  26. Pearse B. M. Assembly of the mannose-6-phosphate receptor into reconstituted clathrin coats. EMBO J. 1985 Oct;4(10):2457–2460. doi: 10.1002/j.1460-2075.1985.tb03956.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Pypaert M., Lucocq J. M., Warren G. Coated pits in interphase and mitotic A431 cells. Eur J Cell Biol. 1987 Dec;45(1):23–29. [PubMed] [Google Scholar]
  31. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Sandvig K., Olsnes S., Petersen O. W., van Deurs B. Acidification of the cytosol inhibits endocytosis from coated pits. J Cell Biol. 1987 Aug;105(2):679–689. doi: 10.1083/jcb.105.2.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sterio D. C. The unbiased estimation of number and sizes of arbitrary particles using the disector. J Microsc. 1984 May;134(Pt 2):127–136. doi: 10.1111/j.1365-2818.1984.tb02501.x. [DOI] [PubMed] [Google Scholar]
  36. Tycko B., Maxfield F. R. Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell. 1982 Mar;28(3):643–651. doi: 10.1016/0092-8674(82)90219-7. [DOI] [PubMed] [Google Scholar]
  37. Warren G., Woodman P., Pypaert M., Smythe E. Cell-free assays and the mechanism of receptor-mediated endocytosis. Trends Biochem Sci. 1988 Dec;13(12):462–465. doi: 10.1016/0968-0004(88)90229-0. [DOI] [PubMed] [Google Scholar]
  38. Woodman P. G., Warren G. Fusion between vesicles from the pathway of receptor-mediated endocytosis in a cell-free system. Eur J Biochem. 1988 Apr 5;173(1):101–108. doi: 10.1111/j.1432-1033.1988.tb13972.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES