100-kDa polypeptides in peripheral clathrin-coated vesicles are required for receptor-mediated endocytosis (original) (raw)

Abstract

The role of the 100-kDa polypeptide components of clathrin-coated vesicles in endocytosis was investigated by microinjection of specific monoclonal antibodies. Receptor-mediated uptake of transferrin and liposomes was quantitatively inhibited. These results show that the 100-kDa polypeptides are directly involved in localized clathrin assembly at the cell periphery and are markers for the endocytic pathway. This demonstrates an in situ function of these polypeptides and the protein complexes in which they are found.

9289

Images in this article

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. Blank G. S., Brodsky F. M. Clathrin assembly involves a light chain-binding region. J Cell Biol. 1987 Nov;105(5):2011–2019. doi: 10.1083/jcb.105.5.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blank G. S., Brodsky F. M. Site-specific disruption of clathrin assembly produces novel structures. EMBO J. 1986 Sep;5(9):2087–2095. doi: 10.1002/j.1460-2075.1986.tb04470.x. [DOI] [PMC free article] [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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Brodsky F. M. Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites. J Cell Biol. 1985 Dec;101(6):2047–2054. doi: 10.1083/jcb.101.6.2047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brodsky F. M. Living with clathrin: its role in intracellular membrane traffic. Science. 1988 Dec 9;242(4884):1396–1402. doi: 10.1126/science.2904698. [DOI] [PubMed] [Google Scholar]
  7. Doxsey S. J., Brodsky F. M., Blank G. S., Helenius A. Inhibition of endocytosis by anti-clathrin antibodies. Cell. 1987 Jul 31;50(3):453–463. doi: 10.1016/0092-8674(87)90499-5. [DOI] [PubMed] [Google Scholar]
  8. Furukawa R., Wampler J. E., Fechheimer M. Measurement of the cytoplasmic pH of Dictyostelium discoideum using a low light level microspectrofluorometer. J Cell Biol. 1988 Dec;107(6 Pt 2):2541–2549. doi: 10.1083/jcb.107.6.2541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Kano K., Fendler J. H. Pyranine as a sensitive pH probe for liposome interiors and surfaces. pH gradients across phospholipid vesicles. Biochim Biophys Acta. 1978 May 18;509(2):289–299. doi: 10.1016/0005-2736(78)90048-2. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Manfredi J. J., Bazari W. L. Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities. J Biol Chem. 1987 Sep 5;262(25):12182–12188. [PubMed] [Google Scholar]
  14. Nermut M. V., Williams L. D., Stamatoglou S. C., Bissell D. M. Ultrastructure of ventral membranes of rat hepatocytes spread on type IV collagen. Eur J Cell Biol. 1986 Oct;42(1):35–44. [PubMed] [Google Scholar]
  15. Nicol A., Nermut M. V. A new type of substratum adhesion structure in NRK cells revealed by correlated interference reflection and electron microscopy. Eur J Cell Biol. 1987 Jun;43(3):348–357. [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. 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]
  18. 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]
  19. 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]
  20. Straubinger R. M., Hong K., Friend D. S., Papahadjopoulos D. Endocytosis of liposomes and intracellular fate of encapsulated molecules: encounter with a low pH compartment after internalization in coated vesicles. Cell. 1983 Apr;32(4):1069–1079. doi: 10.1016/0092-8674(83)90291-x. [DOI] [PubMed] [Google Scholar]
  21. Titus J. A., Haugland R., Sharrow S. O., Segal D. M. Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies. J Immunol Methods. 1982;50(2):193–204. doi: 10.1016/0022-1759(82)90225-3. [DOI] [PubMed] [Google Scholar]
  22. Traganos F., Kimmel M., Bueti C., Darzynkiewicz Z. Effects of inhibition of RNA or protein synthesis on CHO cell cycle progression. J Cell Physiol. 1987 Nov;133(2):277–287. doi: 10.1002/jcp.1041330211. [DOI] [PubMed] [Google Scholar]
  23. Tranquillo R. T., Lauffenburger D. A., Zigmond S. H. A stochastic model for leukocyte random motility and chemotaxis based on receptor binding fluctuations. J Cell Biol. 1988 Feb;106(2):303–309. doi: 10.1083/jcb.106.2.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vigers G. P., Crowther R. A., Pearse B. M. Location of the 100 kd-50 kd accessory proteins in clathrin coats. EMBO J. 1986 Sep;5(9):2079–2085. doi: 10.1002/j.1460-2075.1986.tb04469.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]