Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Nov 1;327(Pt 3):741–746. doi: 10.1042/bj3270741

Identification of cytoskeleton-associated proteins in isolated rat liver endosomes.

A Pol 1, D Ortega 1, C Enrich 1
PMCID: PMC1218852  PMID: 9581551

Abstract

The polypeptides of three highly purified endosomal fractions isolated from the livers of oestradiol-treated rats were analysed by Western blotting, and the amount and distribution of intrinsic and cytoskeletal-associated proteins were quantified and studied. The 'late' endosomes [multivesicular bodies (MVBs)] had the lowest content of cytoskeletal-associated proteins, the most significant being the presence of 25% of the total dynein found in endosomes. The 'early' endosome [compartment of uncoupling receptors and ligands (CURL)] fraction contained kinesin (40% of the total in endosomes), dynein (23%), actin (15%) and tubulin (10%). The receptor-recycling compartment (RRC), also demonstrated to be involved in transcytosis, contained the largest number and enrichment of cytoskeletal proteins: actin (84% of the total in endosomes), alpha-actinin (90%), dynein (52%), tubulin (91%) and kinesin (45%). We also analysed and compared the presence of different endosomal markers such as Rab4, Rab5 and cellubrevin (vesicle soluble NSF attachment protein receptor) in CURL (41%, 15% and 60%) and in RRC (44%, 75% and 30% respectively). Finally, the expression of annexins I, II, IV and VI was studied: annexin I was equally distributed between MVBs and CURL; annexin II was highly enriched in RRC (95%), annexin IV was equally distributed between CURL and RRC, and annexin VI was enriched in CURL (57%). The results indicate that isolated rat liver endosomes contain all the required molecular machinery for the achievement of their role in intracellular trafficking.

Full Text

The Full Text of this article is available as a PDF (297.8 KB).

Selected References

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

  1. Apodaca G., Enrich C., Mostov K. E. The calmodulin antagonist, W-13, alters transcytosis, recycling, and the morphology of the endocytic pathway in Madin-Darby canine kidney cells. J Biol Chem. 1994 Jul 22;269(29):19005–19013. [PubMed] [Google Scholar]
  2. Apodaca G., Katz L. A., Mostov K. E. Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes. J Cell Biol. 1994 Apr;125(1):67–86. doi: 10.1083/jcb.125.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartles J. R., Hubbard A. L. Plasma membrane protein sorting in epithelial cells: do secretory pathways hold the key? Trends Biochem Sci. 1988 May;13(5):181–184. doi: 10.1016/0968-0004(88)90147-8. [DOI] [PubMed] [Google Scholar]
  4. Belcher J. D., Hamilton R. L., Brady S. E., Hornick C. A., Jaeckle S., Schneider W. J., Havel R. J. Isolation and characterization of three endosomal fractions from the liver of estradiol-treated rats. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6785–6789. doi: 10.1073/pnas.84.19.6785. [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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Burgoyne R. D., Clague M. J. Annexins in the endocytic pathway. Trends Biochem Sci. 1994 Jun;19(6):231–232. doi: 10.1016/0968-0004(94)90143-0. [DOI] [PubMed] [Google Scholar]
  7. Burgoyne R. D., Geisow M. J. The annexin family of calcium-binding proteins. Review article. Cell Calcium. 1989 Jan;10(1):1–10. doi: 10.1016/0143-4160(89)90038-9. [DOI] [PubMed] [Google Scholar]
  8. Chao Y. S., Windler E. E., Chen G. C., Havel R. J. Hepatic catabolism of rat and human lipoproteins in rats treated with 17 alpha-ethinyl estradiol. J Biol Chem. 1979 Nov 25;254(22):11360–11366. [PubMed] [Google Scholar]
  9. Chapin S. J., Enrich C., Aroeti B., Havel R. J., Mostov K. E. Calmodulin binds to the basolateral targeting signal of the polymeric immunoglobulin receptor. J Biol Chem. 1996 Jan 19;271(3):1336–1342. doi: 10.1074/jbc.271.3.1336. [DOI] [PubMed] [Google Scholar]
  10. Cole N. B., Lippincott-Schwartz J. Organization of organelles and membrane traffic by microtubules. Curr Opin Cell Biol. 1995 Feb;7(1):55–64. doi: 10.1016/0955-0674(95)80045-x. [DOI] [PubMed] [Google Scholar]
  11. Crompton M. R., Moss S. E., Crumpton M. J. Diversity in the lipocortin/calpactin family. Cell. 1988 Oct 7;55(1):1–3. doi: 10.1016/0092-8674(88)90002-5. [DOI] [PubMed] [Google Scholar]
  12. Crompton M. R., Owens R. J., Totty N. F., Moss S. E., Waterfield M. D., Crumpton M. J. Primary structure of the human, membrane-associated Ca2+-binding protein p68 a novel member of a protein family. EMBO J. 1988 Jan;7(1):21–27. doi: 10.1002/j.1460-2075.1988.tb02779.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Daro E., van der Sluijs P., Galli T., Mellman I. Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9559–9564. doi: 10.1073/pnas.93.18.9559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Durrbach A., Louvard D., Coudrier E. Actin filaments facilitate two steps of endocytosis. J Cell Sci. 1996 Feb;109(Pt 2):457–465. doi: 10.1242/jcs.109.2.457. [DOI] [PubMed] [Google Scholar]
  15. Enrich C., Bachs O., Evans W. H. A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions. Biochem J. 1988 Nov 1;255(3):999–1005. doi: 10.1042/bj2550999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Enrich C., Jäckle S., Havel R. J. The polymeric immunoglobulin receptor is the major calmodulin-binding protein in an endosome fraction from rat liver enriched in recycling receptors. Hepatology. 1996 Jul;24(1):226–232. doi: 10.1002/hep.510240136. [DOI] [PubMed] [Google Scholar]
  17. Enrich C., Tabona P., Evans W. H. A two-dimensional electrophoretic analysis of the proteins and glycoproteins of liver plasma membrane domains and endosomes. Implications for endocytosis and transcytosis. Biochem J. 1990 Oct 1;271(1):171–178. doi: 10.1042/bj2710171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Gorvel J. P., Chavrier P., Zerial M., Gruenberg J. rab5 controls early endosome fusion in vitro. Cell. 1991 Mar 8;64(5):915–925. doi: 10.1016/0092-8674(91)90316-q. [DOI] [PubMed] [Google Scholar]
  21. Gruenberg J., Emans N. Annexins in membrane traffic. Trends Cell Biol. 1993 Jul;3(7):224–227. doi: 10.1016/0962-8924(93)90116-i. [DOI] [PubMed] [Google Scholar]
  22. Gruenberg J., Maxfield F. R. Membrane transport in the endocytic pathway. Curr Opin Cell Biol. 1995 Aug;7(4):552–563. doi: 10.1016/0955-0674(95)80013-1. [DOI] [PubMed] [Google Scholar]
  23. Guerini D., Krebs J., Carafoli E. Stimulation of the purified erythrocyte Ca2+-ATPase by tryptic fragments of calmodulin. J Biol Chem. 1984 Dec 25;259(24):15172–15177. [PubMed] [Google Scholar]
  24. HAVEL R. J., EDER H. A., BRAGDON J. H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955 Sep;34(9):1345–1353. doi: 10.1172/JCI103182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Harder T., Gerke V. The subcellular distribution of early endosomes is affected by the annexin II2p11(2) complex. J Cell Biol. 1993 Dec;123(5):1119–1132. doi: 10.1083/jcb.123.5.1119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hornick C. A., Hamilton R. L., Spaziani E., Enders G. H., Havel R. J. Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus. J Cell Biol. 1985 May;100(5):1558–1569. doi: 10.1083/jcb.100.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jäckle S., Beisiegel U., Rinninger F., Buck F., Grigoleit A., Block A., Gröger I., Greten H., Windler E. Annexin VI, a marker protein of hepatocytic endosomes. J Biol Chem. 1994 Jan 14;269(2):1026–1032. [PubMed] [Google Scholar]
  28. Jäckle S., Runquist E. A., Miranda-Brady S., Havel R. J. Trafficking of the epidermal growth factor receptor and transferrin in three hepatocytic endosomal fractions. J Biol Chem. 1991 Jan 25;266(3):1396–1402. [PubMed] [Google Scholar]
  29. Jäckle S., Runquist E., Brady S., Hamilton R. L., Havel R. J. Isolation and characterization of three endosomal fractions from the liver of normal rats after lipoprotein loading. J Lipid Res. 1991 Mar;32(3):485–498. [PubMed] [Google Scholar]
  30. Kok J. W., Babia T., Hoekstra D. Sorting of sphingolipids in the endocytic pathway of HT29 cells. J Cell Biol. 1991 Jul;114(2):231–239. doi: 10.1083/jcb.114.2.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Lin H. C., Südhof T. C., Anderson R. G. Annexin VI is required for budding of clathrin-coated pits. Cell. 1992 Jul 24;70(2):283–291. doi: 10.1016/0092-8674(92)90102-i. [DOI] [PubMed] [Google Scholar]
  33. Mellman I. Endocytosis and molecular sorting. Annu Rev Cell Dev Biol. 1996;12:575–625. doi: 10.1146/annurev.cellbio.12.1.575. [DOI] [PubMed] [Google Scholar]
  34. Murphy C., Saffrich R., Grummt M., Gournier H., Rybin V., Rubino M., Auvinen P., Lütcke A., Parton R. G., Zerial M. Endosome dynamics regulated by a Rho protein. Nature. 1996 Dec 5;384(6608):427–432. doi: 10.1038/384427a0. [DOI] [PubMed] [Google Scholar]
  35. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  36. Oda H., Stockert R. J., Collins C., Wang H., Novikoff P. M., Satir P., Wolkoff A. W. Interaction of the microtubule cytoskeleton with endocytic vesicles and cytoplasmic dynein in cultured rat hepatocytes. J Biol Chem. 1995 Jun 23;270(25):15242–15249. doi: 10.1074/jbc.270.25.15242. [DOI] [PubMed] [Google Scholar]
  37. Pol A., Ortega D., Enrich C. Identification and distribution of proteins in isolated endosomal fractions of rat liver: involvement in endocytosis, recycling and transcytosis. Biochem J. 1997 Apr 15;323(Pt 2):435–443. doi: 10.1042/bj3230435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Quintart J., Courtoy P. J., Limet J. N., Baudhuin P. Galactose-specific endocytosis in rat liver. Biochemical and morphological characterization of a low-density compartment isolated from hepatocytes. Eur J Biochem. 1983 Mar 1;131(1):105–112. doi: 10.1111/j.1432-1033.1983.tb07236.x. [DOI] [PubMed] [Google Scholar]
  39. Robinson M. S., Watts C., Zerial M. Membrane dynamics in endocytosis. Cell. 1996 Jan 12;84(1):13–21. doi: 10.1016/s0092-8674(00)80988-5. [DOI] [PubMed] [Google Scholar]
  40. Swairjo M. A., Seaton B. A. Annexin structure and membrane interactions: a molecular perspective. Annu Rev Biophys Biomol Struct. 1994;23:193–213. doi: 10.1146/annurev.bb.23.060194.001205. [DOI] [PubMed] [Google Scholar]
  41. de Figueiredo P., Brown W. J. A role for calmodulin in organelle membrane tubulation. Mol Biol Cell. 1995 Jul;6(7):871–887. doi: 10.1091/mbc.6.7.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. van der Sluijs P., Hull M., Webster P., Mâle P., Goud B., Mellman I. The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell. 1992 Sep 4;70(5):729–740. doi: 10.1016/0092-8674(92)90307-x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES