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. 1989 Jun 1;108(6):2093–2099. doi: 10.1083/jcb.108.6.2093

Reconstitution of an endosome-lysosome interaction in a cell-free system

PMCID: PMC2115598  PMID: 2472400

Abstract

A cell-free model for the transfer of endocytosed material to lysosomes is described. Rat liver late endosomes, loaded in vivo with radiolabeled ligand by intravenous injection shortly before killing the animal, showed a specific interaction with lysosomes when incubated at 37 degrees C in the presence of cytosol and an ATP regenerating system. The location of the ligand, generally asialofetuin, was analyzed by isopycnic centrifugation on Nycodenz gradients. Appearance of radiolabel in the lysosomal position on such gradients was maximal after approximately 30 min at 37 degrees C and required the provision of undamaged cytosol, lysosomes, and an ATP regenerating system. It could not be accounted for by nonspecific bulk aggregation of membranes. Transfer occurred only from late endosomes; radiolabel in early endosomes was unaffected. Digestion of the asialofetuin, as shown by the appearance of TCA-soluble radioactivity, occurred on incubation at 37 degrees C and was increased by the provision of an ATP regenerating system.

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Selected References

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  1. 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]
  2. Balch W. E., Rothman J. E. Characterization of protein transport between successive compartments of the Golgi apparatus: asymmetric properties of donor and acceptor activities in a cell-free system. Arch Biochem Biophys. 1985 Jul;240(1):413–425. doi: 10.1016/0003-9861(85)90046-3. [DOI] [PubMed] [Google Scholar]
  3. Berg T., Kindberg G. M., Ford T., Blomhoff R. Intracellular transport of asialoglycoproteins in rat hepatocytes. Evidence for two subpopulations of lysosomes. Exp Cell Res. 1985 Dec;161(2):285–296. doi: 10.1016/0014-4827(85)90086-2. [DOI] [PubMed] [Google Scholar]
  4. Braell W. A. Fusion between endocytic vesicles in a cell-free system. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1137–1141. doi: 10.1073/pnas.84.5.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Branch W. J., Mullock B. M., Luzio J. P. Rapid subcellular fractionation of the rat liver endocytic compartments involved in transcytosis of polymeric immunoglobulin A and endocytosis of asialofetuin. Biochem J. 1987 Jun 1;244(2):311–315. doi: 10.1042/bj2440311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Breitfeld P. P., Simmons C. F., Jr, Strous G. J., Geuze H. J., Schwartz A. L. Cell biology of the asialoglycoprotein receptor system: a model of receptor-mediated endocytosis. Int Rev Cytol. 1985;97:47–95. doi: 10.1016/s0074-7696(08)62348-7. [DOI] [PubMed] [Google Scholar]
  7. Davey J., Hurtley S. M., Warren G. Reconstitution of an endocytic fusion event in a cell-free system. Cell. 1985 Dec;43(3 Pt 2):643–652. doi: 10.1016/0092-8674(85)90236-3. [DOI] [PubMed] [Google Scholar]
  8. Diaz R., Mayorga L., Stahl P. In vitro fusion of endosomes following receptor-mediated endocytosis. J Biol Chem. 1988 May 5;263(13):6093–6100. [PubMed] [Google Scholar]
  9. Drickamer K., Mamon J. F., Binns G., Leung J. O. Primary structure of the rat liver asialoglycoprotein receptor. Structural evidence for multiple polypeptide species. J Biol Chem. 1984 Jan 25;259(2):770–778. [PubMed] [Google Scholar]
  10. Dunn W. A., Hubbard A. L., Aronson N. N., Jr Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of 125I-asialofetuin by the perfused rat liver. J Biol Chem. 1980 Jun 25;255(12):5971–5978. [PubMed] [Google Scholar]
  11. Dunn W. A., LaBadie J. H., Aronson N. N., Jr Inhibition of 125I-asialofetuin catabolism by leupeptin in the perfused rat liver and in vivo. J Biol Chem. 1979 May 25;254(10):4191–4196. [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Geuze H. J., van der Donk H. A., Simmons C. F., Slot J. W., Strous G. J., Schwartz A. L. Receptor-mediated endocytosis in liver parenchymal cells. Int Rev Exp Pathol. 1986;29:113–171. [PubMed] [Google Scholar]
  15. Gruenberg J. E., Howell K. E. Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system. EMBO J. 1986 Dec 1;5(12):3091–3101. doi: 10.1002/j.1460-2075.1986.tb04615.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gruenberg J., Howell K. E. An internalized transmembrane protein resides in a fusion-competent endosome for less than 5 minutes. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5758–5762. doi: 10.1073/pnas.84.16.5758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kindberg G. M., Refsnes M., Christoffersen T., Norum K. R., Berg T. The relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes. J Biol Chem. 1987 May 25;262(15):7066–7071. [PubMed] [Google Scholar]
  18. Luzio J. P., Stanley K. K. The isolation of endosome-derived vesicles from rat hepatocytes. Biochem J. 1983 Oct 15;216(1):27–36. doi: 10.1042/bj2160027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maguire G. A., Docherty K., Hales C. N. Sugar transport in rat liver lysosomes. Direct demonstration by using labelled sugars. Biochem J. 1983 Apr 15;212(1):211–218. doi: 10.1042/bj2120211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Maguire G. A., Luzio J. P. The presence and orientation of ecto-5'-nucleotidase in rat liver lysosomes. FEBS Lett. 1985 Jan 21;180(1):122–126. doi: 10.1016/0014-5793(85)80244-1. [DOI] [PubMed] [Google Scholar]
  21. Malaisse W. J., Hutton J. C., Kawazu S., Sener A. The stimulus-secretion coupling of glucose-induced insulin release. Metabolic effects of menadione in isolated islets. Eur J Biochem. 1978 Jun 1;87(1):121–130. doi: 10.1111/j.1432-1033.1978.tb12357.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Mueller S. C., Hubbard A. L. Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes. J Cell Biol. 1986 Mar;102(3):932–942. doi: 10.1083/jcb.102.3.932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Pearse B. M. Clathrin and coated vesicles. EMBO J. 1987 Sep;6(9):2507–2512. doi: 10.1002/j.1460-2075.1987.tb02536.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Perez J. H., Branch W. J., Smith L., Mullock B. M., Luzio J. P. Investigation of endosomal compartments involved in endocytosis and transcytosis of polymeric immunoglobulin A by subcellular fractionation of perfused isolated rat liver. Biochem J. 1988 May 1;251(3):763–770. doi: 10.1042/bj2510763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Roederer M., Bowser R., Murphy R. F. Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: evidence for a maturation model for the formation of lysosomes. J Cell Physiol. 1987 May;131(2):200–209. doi: 10.1002/jcp.1041310209. [DOI] [PubMed] [Google Scholar]
  28. Rothman J. E. Transport of the vesicular stomatitis glycoprotein to trans Golgi membranes in a cell-free system. J Biol Chem. 1987 Sep 15;262(26):12502–12510. [PubMed] [Google Scholar]
  29. Schmid S. L., Fuchs R., Male P., Mellman I. Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes. Cell. 1988 Jan 15;52(1):73–83. doi: 10.1016/0092-8674(88)90532-6. [DOI] [PubMed] [Google Scholar]
  30. Schwartz A. L., Marshak-Rothstein A., Rup D., Lodish H. F. Identification and quantification of the rat hepatocyte asialoglycoprotein receptor. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3348–3352. doi: 10.1073/pnas.78.6.3348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Steer C. J., Ashwell G. Hepatic membrane receptors for glycoproteins. Prog Liver Dis. 1986;8:99–123. [PubMed] [Google Scholar]
  32. Tolleshaug H., Kolset S. O., Berg T. The influence of cellular ATP levels on receptor-mediated endocytosis and degradation of asialo-glycoproteins in suspended hepatocytes. Biochem Pharmacol. 1985 May 15;34(10):1639–1645. doi: 10.1016/0006-2952(85)90628-8. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Wattiaux R., Wattiaux-De Coninck S., Ronveaux-dupal M. F., Dubois F. Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient. J Cell Biol. 1978 Aug;78(2):349–368. doi: 10.1083/jcb.78.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Woodman P. G., Warren G. Fusion between vesicles from the pathway of receptor-mediated endocytosis in a cell-free system. Eur J Biochem. 1988 Apr 5;173(1):101–108. doi: 10.1111/j.1432-1033.1988.tb13972.x. [DOI] [PubMed] [Google Scholar]

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