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PMID: 1900301 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Isolation of functional, coated, endocytic vesicles.

The Journal of cell biology ·Vol. 112 ·No. 6 ·1991-03-00 ·Pages 1133-41

Woodman PG, Warren G

Abstract

Brief internalization of [125I]transferrin was used to label coated endocytic vesicles, which were then purified using a combination of 2H2O and 2H2O/Ficoll density gradients. Purification was monitored using an assay measuring fusion of endocytic organelles, so as to isolate functional vesicles. Isolated vesicles had all the properties of clathrin-coated vesicles, being enriched for the major components of clathrin coats and uncoated by either 1 M Tris-HCl or an uncoating ATPase. Nearly half of the labeled vesicles were able to participate in subsequent fusion events, as measured by the cell-free assay. Fusion was specific, requiring energy and cytosol, and being sensitive to N-ethyl maleimide.

MeSH Terms
Cell Fractionation/methods Cell Line Centrifugation, Density Gradient/methods Clathrin/isolation & purification,metabolism Coated Pits, Cell-Membrane/metabolism,ultrastructure Deferoxamine/pharmacology Endocytosis Humans Kinetics Membrane Proteins/biosynthesis,isolation & purification Methionine/metabolism Microscopy, Electron Organelles/metabolism,ultrastructure Transferrin/metabolism
Chemicals
Clathrin Membrane Proteins Transferrin Methionine Deferoxamine
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Woodman P G
Cell Biology Laboratory, Imperial Cancer Research Fund, London, England.
Warren G
References (49)
49 references, click to expand
  1. Regulatory role for GTP-binding proteins in endocytosis.
    Science. 1989 Jun 23;244(4911):1475-7 PMID: 2499930
  2. Isolation of a functional vesicular intermediate that mediates ER to Golgi transport in yeast.
    J Cell Biol. 1990 Jul;111(1):45-53 PMID: 2195039
  3. 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 PMID: 3955657
  4. 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):743-52 PMID: 2897244
  5. Reconstitution of an endocytic fusion event in a cell-free system.
    Cell. 1985 Dec;43(3 Pt 2):643-52 PMID: 4075403
  6. Location of the 100 kd-50 kd accessory proteins in clathrin coats.
    EMBO J. 1986 Sep;5(9):2079-85 PMID: 2877872
  7. Enzymatic recycling of clathrin from coated vesicles.
    Cell. 1986 Jul 4;46(1):5-9 PMID: 2872968
  8. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system.
    Annu Rev Cell Biol. 1985;1:1-39 PMID: 2881559
  9. Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system.
    EMBO J. 1986 Dec 1;5(12):3091-101 PMID: 3028771
  10. Uncoating ATPase is a member of the 70 kilodalton family of stress proteins.
    Cell. 1986 Apr 11;45(1):3-13 PMID: 2937542
  11. Cell-free assays and the mechanism of receptor-mediated endocytosis.
    Trends Biochem Sci. 1988 Dec;13(12):462-5 PMID: 3150882
  12. Formation of coated vesicles from coated pits in broken A431 cells.
    J Cell Biol. 1989 Mar;108(3):843-53 PMID: 2564003
  13. Membrane traffic in endocytosis: insights from cell-free assays.
    Annu Rev Cell Biol. 1989;5:453-81 PMID: 2557061
  14. Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro.
    J Cell Biol. 1989 Apr;108(4):1301-16 PMID: 2538480
  15. Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack.
    Cell. 1989 Feb 10;56(3):357-68 PMID: 2536591
  16. Fusion of endocytic vesicles in a cell-free system.
    Methods Cell Biol. 1989;31:197-206 PMID: 2674625
  17. Purification of three related peripheral membrane proteins needed for vesicular transport.
    J Biol Chem. 1990 Jun 15;265(17):10109-17 PMID: 2190980
  18. Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats.
    J Cell Biol. 1983 Nov;97(5 Pt 1):1339-47 PMID: 6138359
  19. An enzyme that removes clathrin coats: purification of an uncoating ATPase.
    J Cell Biol. 1984 Aug;99(2):723-33 PMID: 6146630
  20. Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells.
    J Cell Biol. 1983 Aug;97(2):508-21 PMID: 6309862
  21. Binding of apotransferrin to K562 cells: explanation of the transferrin cycle.
    Proc Natl Acad Sci U S A. 1983 Apr;80(8):2263-6 PMID: 6300904
  22. 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 PMID: 6411734
  23. Three-dimensional visualization of coated vesicle formation in fibroblasts.
    J Cell Biol. 1980 Mar;84(3):560-83 PMID: 6987244
  24. Coated vesicles from human placenta carry ferritin, transferrin, and immunoglobulin G.
    Proc Natl Acad Sci U S A. 1982 Jan;79(2):451-5 PMID: 6952195
  25. Isolation and preliminary characterization of clathrin-associated proteins.
    Eur J Biochem. 1982 Jul;125(2):463-70 PMID: 7117245
  26. Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts.
    Cell. 1977 Mar;10(3):351-64 PMID: 191195
  27. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
    Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4 PMID: 388439
  28. Clathrin-coated vesicles: isolation, dissociation and factor-dependent reassociation of clathrin baskets.
    Cell. 1979 Feb;16(2):303-12 PMID: 455437
  29. 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):849-57 PMID: 637870
  30. 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;72:248-54 PMID: 942051
  31. Coated vesicles from pig brain: purification and biochemical characterization.
    J Mol Biol. 1975 Sep 5;97(1):93-8 PMID: 1177317
  32. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  33. A quantitative analysis of the endocytic pathway in baby hamster kidney cells.
    J Cell Biol. 1989 Dec;109(6 Pt 1):2703-20 PMID: 2592402
  34. Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack.
    Cell. 1989 Jul 28;58(2):329-36 PMID: 2752426
  35. Vesicle fusion following receptor-mediated endocytosis requires a protein active in Golgi transport.
    Nature. 1989 Jun 1;339(6223):398-400 PMID: 2725659
  36. Cell biology: biological cold fusion.
    Nature. 1989 Jun 1;339(6223):336 PMID: 2725657
  37. Structure of human clathrin light chains. Conservation of light chain polymorphism in three mammalian species.
    J Biol Chem. 1988 Nov 15;263(32):16688-95 PMID: 3267234
  38. Release of putative exocytic transport vesicles from perforated MDCK cells.
    EMBO J. 1988 Dec 20;7(13):4075-85 PMID: 3243273
  39. Role of an N-ethylmaleimide-sensitive transport component in promoting fusion of transport vesicles with cisternae of the Golgi stack.
    Cell. 1988 Jul 15;54(2):221-7 PMID: 3390865
  40. Fusion between vesicles from the pathway of receptor-mediated endocytosis in a cell-free system.
    Eur J Biochem. 1988 Apr 5;173(1):101-8 PMID: 3356185
  41. Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes.
    Cell. 1988 Jan 15;52(1):73-83 PMID: 3345561
  42. Purification and properties of 100-kd proteins from coated vesicles and their reconstitution with clathrin.
    EMBO J. 1984 Sep;3(9):1951-7 PMID: 6149117
  43. 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):405-16 PMID: 6498939
  44. Fusion between endocytic vesicles in a cell-free system.
    Proc Natl Acad Sci U S A. 1987 Mar;84(5):1137-41 PMID: 3469657
  45. Assembled and unassembled pools of clathrin: a quantitative study using an enzyme immunoassay.
    J Cell Biol. 1985 Feb;100(2):521-7 PMID: 3968176
  46. Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells.
    J Cell Biol. 1990 Sep;111(3):987-1000 PMID: 2202740
  47. Dissociation of clathrin from coated vesicles by the uncoating ATPase.
    J Biol Chem. 1990 Apr 25;265(12):6682-7 PMID: 1969864
  48. SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast.
    Cell. 1990 May 18;61(4):709-21 PMID: 2111733
  49. Receptors compete for adaptors found in plasma membrane coated pits.
    EMBO J. 1988 Nov;7(11):3331-6 PMID: 2905261
Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.112.6.1133
Published
1991-03-00
Pages
1133-41
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2288903
Subset
IM
Grants
Wellcome Trust · United Kingdom
External Links
PubMed source DOI: 10.1083/jcb.112.6.1133 PMC Full Text View journal details
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