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. 1985 May 1;100(5):1558–1569. doi: 10.1083/jcb.100.5.1558

Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus

PMCID: PMC2113894  PMID: 3988801

Abstract

Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor- mediated endocytosis. MVBs also contained numerous small vesicles, 0.05- 0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins.

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Selected References

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  1. Ahlberg J., Marzella L., Glaumann H. Uptake and degradation of proteins by isolated rat liver lysosomes. Suggestion of a microautophagic pathway of proteolysis. Lab Invest. 1982 Dec;47(6):523–532. [PubMed] [Google Scholar]
  2. Altstiel L., Branton D. Fusion of coated vesicles with lysosomes: measurement with a fluorescence assay. Cell. 1983 Mar;32(3):921–929. doi: 10.1016/0092-8674(83)90077-6. [DOI] [PubMed] [Google Scholar]
  3. Bergeron J. J., Resch L., Rachubinski R., Patel B. A., Posner B. I. Effect of colchicine on internalization of prolactin in female rat liver: an in vivo radioautographic study. J Cell Biol. 1983 Mar;96(3):875–886. doi: 10.1083/jcb.96.3.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown M. S., Anderson R. G., Goldstein J. L. Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell. 1983 Mar;32(3):663–667. doi: 10.1016/0092-8674(83)90052-1. [DOI] [PubMed] [Google Scholar]
  5. Carpentier J. L., Gorden P., Freychet P., Le Cam A., Orci L. Lysosomal association of internalized 125I-insulin in isolated rat hepatocytes. Direct demonstration by quantitative electron microscopic autoradiography. J Clin Invest. 1979 Jun;63(6):1249–1261. doi: 10.1172/JCI109420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carpentier J. L., Gorden P., Goldstein J. L., Anderson R. G., Brown M. S., Orci L. Binding and internalization of 125I-LDL in normal and mutant human fibroblasts. A quantitative autoradiographic study. Exp Cell Res. 1979 Jun;121(1):135–142. doi: 10.1016/0014-4827(79)90453-1. [DOI] [PubMed] [Google Scholar]
  7. Chao Y. S., Jones A. L., Hradek G. T., Windler E. E., Havel R. J. Autoradiographic localization of the sites of uptake, cellular transport, and catabolism of low density lipoproteins in the liver of normal and estrogen-treated rats. Proc Natl Acad Sci U S A. 1981 Jan;78(1):597–601. doi: 10.1073/pnas.78.1.597. [DOI] [PMC free article] [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. 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]
  10. Dickson R. B., Beguinot L., Hanover J. A., Richert N. D., Willingham M. C., Pastan I. Isolation and characterization of a highly enriched preparation of receptosomes (endosomes) from a human cell line. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5335–5339. doi: 10.1073/pnas.80.17.5335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dickson R. B., Hanover J. A., Willingham M. C., Pastan I. Prelysosomal divergence of transferrin and epidermal growth factor during receptor-mediated endocytosis. Biochemistry. 1983 Nov 22;22(24):5667–5674. doi: 10.1021/bi00293a033. [DOI] [PubMed] [Google Scholar]
  12. Ehrenreich J. H., Bergeron J. J., Siekevitz P., Palade G. E. Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J Cell Biol. 1973 Oct;59(1):45–72. doi: 10.1083/jcb.59.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Elias P. M., Goerke J., Friend D. S., Brown B. E. Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes. J Cell Biol. 1978 Aug;78(2):577–596. doi: 10.1083/jcb.78.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Farquhar M. G. Multiple pathways of exocytosis, endocytosis, and membrane recycling: validation of a Golgi route. Fed Proc. 1983 May 15;42(8):2407–2413. [PubMed] [Google Scholar]
  15. Felker T. E., Fainaru M., Hamilton R. L., Havel R. J. Secretion of the arginine-rich and A-I apolipoproteins by the isolated perfused rat liver. J Lipid Res. 1977 Jul;18(4):465–473. [PubMed] [Google Scholar]
  16. Friend D. S., Farquhar M. G. Functions of coated vesicles during protein absorption in the rat vas deferens. J Cell Biol. 1967 Nov;35(2):357–376. doi: 10.1083/jcb.35.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Geuze H. J., Slot J. W., Strous G. J., Schwartz A. L. The pathway of the asialoglycoprotein-ligand during receptor-mediated endocytosis: a morphological study with colloidal gold/ligand in the human hepatoma cell line, Hep G2. Eur J Cell Biol. 1983 Nov;32(1):38–44. [PubMed] [Google Scholar]
  20. Goldstein J. L., Anderson R. G., Brown M. S. Receptor-mediated endocytosis and the cellular uptake of low density lipoprotein. Ciba Found Symp. 1982;(92):77–95. doi: 10.1002/9780470720745.ch5. [DOI] [PubMed] [Google Scholar]
  21. Gorden P., Carpentier J. L., Cohen S., Orci L. Epidermal growth factor: morphological demonstration of binding, internalization, and lysosomal association in human fibroblasts. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5025–5029. doi: 10.1073/pnas.75.10.5025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gorden P., Carpentier J. L., Fan J. Y., Orci L. Receptor mediated endocytosis of polypeptide hormones: mechanism and significance. Metabolism. 1982 Jul;31(7):664–669. doi: 10.1016/0026-0495(82)90196-2. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Hamilton R. L., Jr, Goerke J., Guo L. S., Williams M. C., Havel R. J. Unilamellar liposomes made with the French pressure cell: a simple preparative and semiquantitative technique. J Lipid Res. 1980 Nov;21(8):981–992. [PubMed] [Google Scholar]
  25. Hamilton R. L., Williams M. C., Fielding C. J., Havel R. J. Discoidal bilayer structure of nascent high density lipoproteins from perfused rat liver. J Clin Invest. 1976 Sep;58(3):667–680. doi: 10.1172/JCI108513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Howell K. E., Palade G. E. Hepatic Golgi fractions resolved into membrane and content subfractions. J Cell Biol. 1982 Mar;92(3):822–832. doi: 10.1083/jcb.92.3.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Huang H., Kauan J. W., Guilbault G. G. Fluorometric enzymatic determination of total cholesterol in serum. Clin Chem. 1975 Oct;21(11):1605–1608. [PubMed] [Google Scholar]
  28. Jones A. L., Hradek G. T., Hornick C., Renaud G., Windler E. E., Havel R. J. Uptake and processing of remnants of chylomicrons and very low density lipoproteins by rat liver. J Lipid Res. 1984 Nov;25(11):1151–1158. [PubMed] [Google Scholar]
  29. Kane J. P., Hardman D. A., Paulus H. E. Heterogeneity of apolipoprotein B: isolation of a new species from human chylomicrons. Proc Natl Acad Sci U S A. 1980 May;77(5):2465–2469. doi: 10.1073/pnas.77.5.2465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kovanen P. T., Brown M. S., Goldstein J. L. Increased binding of low density lipoprotein to liver membranes from rats treated with 17 alpha-ethinyl estradiol. J Biol Chem. 1979 Nov 25;254(22):11367–11373. [PubMed] [Google Scholar]
  31. Kremmer T., Wisher M. H., Evans W. H. The lipid composition of plasma membrane subfractions originating from the three major functional domains of the rat hepatocyte cell surface. Biochim Biophys Acta. 1976 Dec 14;455(3):655–664. doi: 10.1016/0005-2736(76)90039-0. [DOI] [PubMed] [Google Scholar]
  32. Kruth H. S., Blanchette-Mackie J., Avigan J., Gamble W., Vaughan M. Subcellular localization and quantification of cholesterol in cultured human fibroblasts exposed to human low density lipoprotein. J Lipid Res. 1982 Nov;23(8):1128–1135. [PubMed] [Google Scholar]
  33. 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]
  34. Marzella L., Ahlberg J., Glaumann H. In vitro uptake of particles by lysosomes. Exp Cell Res. 1980 Oct;129(2):460–466. doi: 10.1016/0014-4827(80)90515-7. [DOI] [PubMed] [Google Scholar]
  35. McGookey D. J., Fagerberg K., Anderson R. G. Filipin-cholesterol complexes form in uncoated vesicle membrane derived from coated vesicles during receptor-mediated endocytosis of low density lipoprotein. J Cell Biol. 1983 May;96(5):1273–1278. doi: 10.1083/jcb.96.5.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McKanna J. A., Haigler H. T., Cohen S. Hormone receptor topology and dynamics: morphological analysis using ferritin-labeled epidermal growth factor. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5689–5693. doi: 10.1073/pnas.76.11.5689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Meier P. J., Sztul E. S., Reuben A., Boyer J. L. Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver. J Cell Biol. 1984 Mar;98(3):991–1000. doi: 10.1083/jcb.98.3.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Merion M., Sly W. S. The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts. J Cell Biol. 1983 Mar;96(3):644–650. doi: 10.1083/jcb.96.3.644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Merritt W. D., Morré D. J. A glycosyl transferase of high specific activity in secretory vesicles from isolated Golgi apparatus of rat liver. Biochim Biophys Acta. 1973 Apr 28;304(2):397–407. doi: 10.1016/0304-4165(73)90259-6. [DOI] [PubMed] [Google Scholar]
  40. Miskimins W. K., Shimizu N. Dual pathways for epidermal growth factor processing after receptor-mediated endocytosis. J Cell Physiol. 1982 Sep;112(3):327–338. doi: 10.1002/jcp.1041120304. [DOI] [PubMed] [Google Scholar]
  41. Montesano R., Perrelet A., Vassalli P., Orci L. Absence of filipin-sterol complexes from large coated pits on the surface of culture cells. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6391–6395. doi: 10.1073/pnas.76.12.6391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Morré D. J., Hamilton R. L., Mollenhauer H. H., Mahley R. W., Cunningham W. P., Cheetham R. D., Lequire V. S. Isolation of a Golgi apparatus-rich fraction from rat liver. I. Method and morphology. J Cell Biol. 1970 Mar;44(3):484–491. doi: 10.1083/jcb.44.3.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Novikoff P. M., Yam A. Sites of lipoprotein particles in normal rat hepatocytes. J Cell Biol. 1978 Jan;76(1):1–11. doi: 10.1083/jcb.76.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ohkuma S., Moriyama Y., Takano T. Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence. Proc Natl Acad Sci U S A. 1982 May;79(9):2758–2762. doi: 10.1073/pnas.79.9.2758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Orange R. P., Moore E. G. Functional characterization of rat mast cell arylsulfatase activity. J Immunol. 1976 Dec;117(6):2191–2196. [PubMed] [Google Scholar]
  46. Orci L., Montesano R., Meda P., Malaisse-Lagae F., Brown D., Perrelet A., Vassalli P. Heterogeneous distribution of filipin--cholesterol complexes across the cisternae of the Golgi apparatus. Proc Natl Acad Sci U S A. 1981 Jan;78(1):293–297. doi: 10.1073/pnas.78.1.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pagnan A., Havel R. J., Kane J. P., Kotite L. Characterization of human very low density lipoproteins containing two electrophoretic populations: double pre-beta lipoproteinemia and primary dysbetalipoproteinemia. J Lipid Res. 1977 Sep;18(5):613–622. [PubMed] [Google Scholar]
  48. Pertoft H., Wärmegård B., Hök M. Heterogeneity of lysosomes originating from rat liver parenchymal cells. Metabolic relationship of subpopulations separated by density-gradient centrifugation. Biochem J. 1978 Jul 15;174(1):309–317. doi: 10.1042/bj1740309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  50. Posner B. I., Bergeron J. J., Josefsberg Z., Khan M. N., Khan R. J., Patel B. A., Sikstrom R. A., Verma A. K. Polypeptide hormones: intracellular receptors and internalization. Recent Prog Horm Res. 1981;37:539–582. doi: 10.1016/b978-0-12-571137-1.50016-5. [DOI] [PubMed] [Google Scholar]
  51. Quintart J., Courtoy P. J., Baudhuin P. Receptor-mediated endocytosis in rat liver: purification and enzymic characterization of low density organelles involved in uptake of galactose-exposing proteins. J Cell Biol. 1984 Mar;98(3):877–884. doi: 10.1083/jcb.98.3.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Rome L. H., Garvin A. J., Allietta M. M., Neufeld E. F. Two species of lysosomal organelles in cultured human fibroblasts. Cell. 1979 May;17(1):143–153. doi: 10.1016/0092-8674(79)90302-7. [DOI] [PubMed] [Google Scholar]
  54. Smith G. D., Peters T. J. The localization in rat liver of alkaline phosphodiesterase to a discrete organelle implicated in ligand internalization. Biochim Biophys Acta. 1982 May 5;716(1):24–30. doi: 10.1016/0304-4165(82)90198-2. [DOI] [PubMed] [Google Scholar]
  55. Smith R. M., Jarett L. Ultrastructural basis for chloroquine-induced increase in intracellular insulin in adipocytes: alteration of lysosomal function. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7302–7306. doi: 10.1073/pnas.79.23.7302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Stalenhoef A. F., Malloy M. J., Kane J. P., Havel R. J. Metabolism of apolipoproteins B-48 and B-100 of triglyceride-rich lipoproteins in normal and lipoprotein lipase-deficient humans. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1839–1843. doi: 10.1073/pnas.81.6.1839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Stewart C. P., Hendry E. B. The phospholipins of blood. Biochem J. 1935 Jul;29(7):1683–1689. doi: 10.1042/bj0291683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Stockert R. J., Haimes H. B., Morell A. G., Novikoff P. M., Novikoff A. B., Quintana N., Sternlieb I. Endocytosis of asialoglycoprotein-enzyme conjugates by hepatocytes. Lab Invest. 1980 Dec;43(6):556–563. [PubMed] [Google Scholar]
  59. Storrie B., Pool R. R., Jr, Sachdeva M., Maurey K. M., Oliver C. Evidence for both prelysosomal and lysosomal intermediates in endocytic pathways. J Cell Biol. 1984 Jan;98(1):108–115. doi: 10.1083/jcb.98.1.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Swift L. L., Manowitz N. R., Dunn G. D., LeQuire V. S. Isolation and characterization of hepatic Golgi lipoproteins from hypercholesterolemic rats. J Clin Invest. 1980 Sep;66(3):415–425. doi: 10.1172/JCI109871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. 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]
  62. Wall D. A., Wilson G., Hubbard A. L. The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell. 1980 Aug;21(1):79–93. doi: 10.1016/0092-8674(80)90116-6. [DOI] [PubMed] [Google Scholar]
  63. Whetton A. D., Houslay M. D., Dodd N. J., Evans W. H. The lipid fluidity of rat liver membrane subfractions. Biochem J. 1983 Sep 15;214(3):851–854. doi: 10.1042/bj2140851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Willingham M. C., Pastan I. H. Transit of epidermal growth factor through coated pits of the Golgi system. J Cell Biol. 1982 Jul;94(1):207–212. doi: 10.1083/jcb.94.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Windler E. E., Kovanen P. T., Chao Y. S., Brown M. S., Havel R. J., Goldstein J. L. The estradiol-stimulated lipoprotein receptor of rat liver. A binding site that membrane mediates the uptake of rat lipoproteins containing apoproteins B and E. J Biol Chem. 1980 Nov 10;255(21):10464–10471. [PubMed] [Google Scholar]
  66. Zannis V. I., Breslow J. L., Utermann G., Mahley R. W., Weisgraber K. H., Havel R. J., Goldstein J. L., Brown M. S., Schonfeld G., Hazzard W. R. Proposed nomenclature of apoE isoproteins, apoE genotypes, and phenotypes. J Lipid Res. 1982 Aug;23(6):911–914. [PubMed] [Google Scholar]
  67. van't Hooft F., Havel R. J. Metabolism of chromatographically separated rat serum lipoproteins specifically labeled with 125I-apolipoprotein E. J Biol Chem. 1981 Apr 25;256(8):3963–3968. [PubMed] [Google Scholar]

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