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. 1996 Jan 1;132(1):21–33. doi: 10.1083/jcb.132.1.21

A novel class of clathrin-coated vesicles budding from endosomes

PMCID: PMC2120710  PMID: 8567724

Abstract

Clathrin-coated vesicles transport selective integral membrane proteins from the plasma membrane to endosomes and from the TGN to endosomes. Recycling of proteins from endosomes to the plasma membrane occurs via unidentified vesicles. To study this pathway, we used a novel technique that allows for the immunoelectron microscopic examination of transferrin receptor-containing endosomes in nonsectioned cells. Endosomes were identified as separate discontinuous tubular-vesicular entities. Each endosome was decorated, mainly on the tubules, with many clathrin-coated buds. Endosome-associated clathrin-coated buds were discerned from plasma membrane-derived clathrin-coated vesicles by three criteria: size (60 nm and 100 nm, respectively), continuity with endosomes, and the lack of labeling for alpha-adaptin. They were also distinguished from TGN-derived clathrin-coated vesicles by their location at the periphery of the cell, size, and the lack of labeling for gamma-adaptin. In the presence of brefeldin A, a large continuous endosomal network was formed. Transferrin receptor recycling as well as the formation of clathrin-coated pits at endosomes was inhibited in the presence of brefeldin A. Together with the localization of transferrin receptors at endosome-associated buds, this indicates that a novel class of clathrin-coated vesicles serves an exit pathway from endosomes. The target organelles for endosome-derived clathrin-coated vesicles remain, however, to be identified.

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Selected References

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  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. Allen R. D., Schroeder C. C., Fok A. K. Endosomal system of Paramecium: coated pits to early endosomes. J Cell Sci. 1992 Feb;101(Pt 2):449–461. doi: 10.1242/jcs.101.2.449. [DOI] [PubMed] [Google Scholar]
  3. Barlowe C., Orci L., Yeung T., Hosobuchi M., Hamamoto S., Salama N., Rexach M. F., Ravazzola M., Amherdt M., Schekman R. COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell. 1994 Jun 17;77(6):895–907. doi: 10.1016/0092-8674(94)90138-4. [DOI] [PubMed] [Google Scholar]
  4. Beck K. A., Chang M., Brodsky F. M., Keen J. H. Clathrin assembly protein AP-2 induces aggregation of membrane vesicles: a possible role for AP-2 in endosome formation. J Cell Biol. 1992 Nov;119(4):787–796. doi: 10.1083/jcb.119.4.787. [DOI] [PMC free article] [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. Chin D. J., Straubinger R. M., Acton S., Näthke I., Brodsky F. M. 100-kDa polypeptides in peripheral clathrin-coated vesicles are required for receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9289–9293. doi: 10.1073/pnas.86.23.9289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Connolly C. N., Futter C. E., Gibson A., Hopkins C. R., Cutler D. F. Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase. J Cell Biol. 1994 Nov;127(3):641–652. doi: 10.1083/jcb.127.3.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Courtoy P. J., Quintart J., Baudhuin P. Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles. J Cell Biol. 1984 Mar;98(3):870–876. doi: 10.1083/jcb.98.3.870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. D'Souza-Schorey C., Li G., Colombo M. I., Stahl P. D. A regulatory role for ARF6 in receptor-mediated endocytosis. Science. 1995 Feb 24;267(5201):1175–1178. doi: 10.1126/science.7855600. [DOI] [PubMed] [Google Scholar]
  10. DOURMASHKIN R. R., DOUGHERTY R. M., HARRIS R. J. Electron microscopic observations on Rous sarcoma virus and cell membranes. Nature. 1962 Jun 23;194:1116–1119. doi: 10.1038/1941116a0. [DOI] [PubMed] [Google Scholar]
  11. Damke H., Klumperman J., von Figura K., Braulke T. Effects of brefeldin A on the endocytic route. Redistribution of mannose 6-phosphate/insulin-like growth factor II receptors to the cell surface. J Biol Chem. 1991 Dec 25;266(36):24829–24833. [PubMed] [Google Scholar]
  12. Doms R. W., Russ G., Yewdell J. W. Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J Cell Biol. 1989 Jul;109(1):61–72. doi: 10.1083/jcb.109.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Draper R. K., Goda Y., Brodsky F. M., Pfeffer S. R. Antibodies to clathrin inhibit endocytosis but not recycling to the trans Golgi network in vitro. Science. 1990 Jun 22;248(4962):1539–1541. doi: 10.1126/science.2163108. [DOI] [PubMed] [Google Scholar]
  14. Felder S., Miller K., Moehren G., Ullrich A., Schlessinger J., Hopkins C. R. Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body. Cell. 1990 May 18;61(4):623–634. doi: 10.1016/0092-8674(90)90474-s. [DOI] [PubMed] [Google Scholar]
  15. Fujiwara T., Oda K., Yokota S., Takatsuki A., Ikehara Y. Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum. J Biol Chem. 1988 Dec 5;263(34):18545–18552. [PubMed] [Google Scholar]
  16. Fukuda M. Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking. J Biol Chem. 1991 Nov 15;266(32):21327–21330. [PubMed] [Google Scholar]
  17. Futter C. E., Felder S., Schlessinger J., Ullrich A., Hopkins C. R. Annexin I is phosphorylated in the multivesicular body during the processing of the epidermal growth factor receptor. J Cell Biol. 1993 Jan;120(1):77–83. doi: 10.1083/jcb.120.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Geuze H. J., Morré D. J. Trans-Golgi reticulum. J Electron Microsc Tech. 1991 Jan;17(1):24–34. doi: 10.1002/jemt.1060170105. [DOI] [PubMed] [Google Scholar]
  19. Geuze H. J., Slot J. W., Schwartz A. L. Membranes of sorting organelles display lateral heterogeneity in receptor distribution. J Cell Biol. 1987 Jun;104(6):1715–1723. doi: 10.1083/jcb.104.6.1715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Geuze H. J., Slot J. W., Strous G. J., Lodish H. F., Schwartz A. L. Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell. 1983 Jan;32(1):277–287. doi: 10.1016/0092-8674(83)90518-4. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Ghosh R. N., Maxfield F. R. Evidence for nonvectorial, retrograde transferrin trafficking in the early endosomes of HEp2 cells. J Cell Biol. 1995 Feb;128(4):549–561. doi: 10.1083/jcb.128.4.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gruenberg J., Howell K. E. Membrane traffic in endocytosis: insights from cell-free assays. Annu Rev Cell Biol. 1989;5:453–481. doi: 10.1146/annurev.cb.05.110189.002321. [DOI] [PubMed] [Google Scholar]
  24. Guarnieri F. G., Arterburn L. M., Penno M. B., Cha Y., August J. T. The motif Tyr-X-X-hydrophobic residue mediates lysosomal membrane targeting of lysosome-associated membrane protein 1. J Biol Chem. 1993 Jan 25;268(3):1941–1946. [PubMed] [Google Scholar]
  25. Hopkins C. R., Gibson A., Shipman M., Miller K. Movement of internalized ligand-receptor complexes along a continuous endosomal reticulum. Nature. 1990 Jul 26;346(6282):335–339. doi: 10.1038/346335a0. [DOI] [PubMed] [Google Scholar]
  26. Hopkins C. R., Gibson A., Shipman M., Strickland D. K., Trowbridge I. S. In migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma membrane of the leading lamella. J Cell Biol. 1994 Jun;125(6):1265–1274. doi: 10.1083/jcb.125.6.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Hunziker W., Whitney J. A., Mellman I. Selective inhibition of transcytosis by brefeldin A in MDCK cells. Cell. 1991 Nov 1;67(3):617–627. doi: 10.1016/0092-8674(91)90535-7. [DOI] [PubMed] [Google Scholar]
  29. Keen J. H. Clathrin and associated assembly and disassembly proteins. Annu Rev Biochem. 1990;59:415–438. doi: 10.1146/annurev.bi.59.070190.002215. [DOI] [PubMed] [Google Scholar]
  30. Killisch I., Steinlein P., Römisch K., Hollinshead R., Beug H., Griffiths G. Characterization of early and late endocytic compartments of the transferrin cycle. Transferrin receptor antibody blocks erythroid differentiation by trapping the receptor in the early endosome. J Cell Sci. 1992 Sep;103(Pt 1):211–232. doi: 10.1242/jcs.103.1.211. [DOI] [PubMed] [Google Scholar]
  31. Klumperman J., Hille A., Veenendaal T., Oorschot V., Stoorvogel W., von Figura K., Geuze H. J. Differences in the endosomal distributions of the two mannose 6-phosphate receptors. J Cell Biol. 1993 Jun;121(5):997–1010. doi: 10.1083/jcb.121.5.997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Le Borgne R., Schmidt A., Mauxion F., Griffiths G., Hoflack B. Binding of AP-1 Golgi adaptors to membranes requires phosphorylated cytoplasmic domains of the mannose 6-phosphate/insulin-like growth factor II receptor. J Biol Chem. 1993 Oct 25;268(30):22552–22556. [PubMed] [Google Scholar]
  33. Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lippincott-Schwartz J., Yuan L., Tipper C., Amherdt M., Orci L., Klausner R. D. Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell. 1991 Nov 1;67(3):601–616. doi: 10.1016/0092-8674(91)90534-6. [DOI] [PubMed] [Google Scholar]
  35. Luzio J. P., Banting G. Eukaryotic membrane traffic: retrieval and retention mechanisms to achieve organelle residence. Trends Biochem Sci. 1993 Oct;18(10):395–398. doi: 10.1016/0968-0004(93)90097-7. [DOI] [PubMed] [Google Scholar]
  36. Mahaffey D. T., Peeler J. S., Brodsky F. M., Anderson R. G. Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity. J Biol Chem. 1990 Sep 25;265(27):16514–16520. [PubMed] [Google Scholar]
  37. Marsh M., Griffiths G., Dean G. E., Mellman I., Helenius A. Three-dimensional structure of endosomes in BHK-21 cells. Proc Natl Acad Sci U S A. 1986 May;83(9):2899–2903. doi: 10.1073/pnas.83.9.2899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Morris S. A., Schröder S., Plessmann U., Weber K., Ungewickell E. Clathrin assembly protein AP180: primary structure, domain organization and identification of a clathrin binding site. EMBO J. 1993 Feb;12(2):667–675. doi: 10.1002/j.1460-2075.1993.tb05700.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Näthke I. S., Heuser J., Lupas A., Stock J., Turck C. W., Brodsky F. M. Folding and trimerization of clathrin subunits at the triskelion hub. Cell. 1992 Mar 6;68(5):899–910. doi: 10.1016/0092-8674(92)90033-9. [DOI] [PubMed] [Google Scholar]
  41. Padh H., Ha J., Lavasa M., Steck T. L. A post-lysosomal compartment in Dictyostelium discoideum. J Biol Chem. 1993 Mar 25;268(9):6742–6747. [PubMed] [Google Scholar]
  42. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  43. Peters P. J., Hsu V. W., Ooi C. E., Finazzi D., Teal S. B., Oorschot V., Donaldson J. G., Klausner R. D. Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments. J Cell Biol. 1995 Mar;128(6):1003–1017. doi: 10.1083/jcb.128.6.1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pevsner J., Volknandt W., Wong B. R., Scheller R. H. Two rat homologs of clathrin-associated adaptor proteins. Gene. 1994 Sep 2;146(2):279–283. doi: 10.1016/0378-1119(94)90306-9. [DOI] [PubMed] [Google Scholar]
  45. Rabinowitz S., Horstmann H., Gordon S., Griffiths G. Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages. J Cell Biol. 1992 Jan;116(1):95–112. doi: 10.1083/jcb.116.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Randazzo P. A., Yang Y. C., Rulka C., Kahn R. A. Activation of ADP-ribosylation factor by Golgi membranes. Evidence for a brefeldin A- and protease-sensitive activating factor on Golgi membranes. J Biol Chem. 1993 May 5;268(13):9555–9563. [PubMed] [Google Scholar]
  47. Reaves B., Banting G. Overexpression of TGN38/41 leads to mislocalisation of gamma-adaptin. FEBS Lett. 1994 Sep 12;351(3):448–456. doi: 10.1016/0014-5793(94)00813-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Riederer M. A., Soldati T., Shapiro A. D., Lin J., Pfeffer S. R. Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network. J Cell Biol. 1994 May;125(3):573–582. doi: 10.1083/jcb.125.3.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rijnboutt S., Aerts H. M., Geuze H. J., Tager J. M., Strous G. J. Mannose 6-phosphate-independent membrane association of cathepsin D, glucocerebrosidase, and sphingolipid-activating protein in HepG2 cells. J Biol Chem. 1991 Mar 15;266(8):4862–4868. [PubMed] [Google Scholar]
  50. Rijnboutt S., Stoorvogel W., Geuze H. J., Strous G. J. Identification of subcellular compartments involved in biosynthetic processing of cathepsin D. J Biol Chem. 1992 Aug 5;267(22):15665–15672. [PubMed] [Google Scholar]
  51. Robinson J. M., Karnovsky M. J. Rapid-freezing cytochemistry: preservation of tubular lysosomes and enzyme activity. J Histochem Cytochem. 1991 Jun;39(6):787–792. doi: 10.1177/39.6.2033237. [DOI] [PubMed] [Google Scholar]
  52. Robinson M. S. The role of clathrin, adaptors and dynamin in endocytosis. Curr Opin Cell Biol. 1994 Aug;6(4):538–544. doi: 10.1016/0955-0674(94)90074-4. [DOI] [PubMed] [Google Scholar]
  53. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  54. Ruscetti T., Cardelli J. A., Niswonger M. L., O'Halloran T. J. Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum. J Cell Biol. 1994 Jul;126(2):343–352. doi: 10.1083/jcb.126.2.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sandoval I. V., Bakke O. Targeting of membrane proteins to endosomes and lysosomes. Trends Cell Biol. 1994 Aug;4(8):292–297. doi: 10.1016/0962-8924(94)90220-8. [DOI] [PubMed] [Google Scholar]
  56. Schmid S. L., Smythe E. Stage-specific assays for coated pit formation and coated vesicle budding in vitro. J Cell Biol. 1991 Sep;114(5):869–880. doi: 10.1083/jcb.114.5.869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Schonhorn J. E., Wessling-Resnick M. Brefeldin A down-regulates the transferrin receptor in K562 cells. Mol Cell Biochem. 1994 Jun 29;135(2):159–169. doi: 10.1007/BF00926519. [DOI] [PubMed] [Google Scholar]
  58. Seaman M. N., Ball C. L., Robinson M. S. Targeting and mistargeting of plasma membrane adaptors in vitro. J Cell Biol. 1993 Dec;123(5):1093–1105. doi: 10.1083/jcb.123.5.1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Slot J. W., Geuze H. J., Gigengack S., Lienhard G. E., James D. E. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol. 1991 Apr;113(1):123–135. doi: 10.1083/jcb.113.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Stamnes M. A., Rothman J. E. The binding of AP-1 clathrin adaptor particles to Golgi membranes requires ADP-ribosylation factor, a small GTP-binding protein. Cell. 1993 Jun 4;73(5):999–1005. doi: 10.1016/0092-8674(93)90277-w. [DOI] [PubMed] [Google Scholar]
  61. Stoorvogel W., Geuze H. J., Griffith J. M., Schwartz A. L., Strous G. J. Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells. J Cell Biol. 1989 Jun;108(6):2137–2148. doi: 10.1083/jcb.108.6.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Stoorvogel W., Geuze H. J., Griffith J. M., Strous G. J. The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum. J Cell Biol. 1988 Jun;106(6):1821–1829. doi: 10.1083/jcb.106.6.1821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Stoorvogel W., Geuze H. J., Strous G. J. Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells. J Cell Biol. 1987 May;104(5):1261–1268. doi: 10.1083/jcb.104.5.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Stoorvogel W., Schwartz A. L., Strous G. J., Fallon R. J. A pool of intracellular phosphorylated asialoglycoprotein receptors which is not involved in endocytosis. J Biol Chem. 1991 Mar 25;266(9):5438–5444. [PubMed] [Google Scholar]
  65. Stoorvogel W., Strous G. J., Geuze H. J., Oorschot V., Schwartz A. L. Late endosomes derive from early endosomes by maturation. Cell. 1991 May 3;65(3):417–427. doi: 10.1016/0092-8674(91)90459-c. [DOI] [PubMed] [Google Scholar]
  66. Strous G. J., van Kerkhof P., van Meer G., Rijnboutt S., Stoorvogel W. Differential effects of brefeldin A on transport of secretory and lysosomal proteins. J Biol Chem. 1993 Feb 5;268(4):2341–2347. [PubMed] [Google Scholar]
  67. Tooze J., Hollinshead M. Tubular early endosomal networks in AtT20 and other cells. J Cell Biol. 1991 Nov;115(3):635–653. doi: 10.1083/jcb.115.3.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Traub L. M., Ostrom J. A., Kornfeld S. Biochemical dissection of AP-1 recruitment onto Golgi membranes. J Cell Biol. 1993 Nov;123(3):561–573. doi: 10.1083/jcb.123.3.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Trowbridge I. S., Collawn J. F., Hopkins C. R. Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol. 1993;9:129–161. doi: 10.1146/annurev.cb.09.110193.001021. [DOI] [PubMed] [Google Scholar]
  70. Wang L. H., Rothberg K. G., Anderson R. G. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J Cell Biol. 1993 Dec;123(5):1107–1117. doi: 10.1083/jcb.123.5.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Wong S. H., Hong W. The SXYQRL sequence in the cytoplasmic domain of TGN38 plays a major role in trans-Golgi network localization. J Biol Chem. 1993 Oct 25;268(30):22853–22862. [PubMed] [Google Scholar]
  72. Wood S. A., Park J. E., Brown W. J. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell. 1991 Nov 1;67(3):591–600. doi: 10.1016/0092-8674(91)90533-5. [DOI] [PubMed] [Google Scholar]

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