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. 1991 Dec 2;115(6):1561–1572. doi: 10.1083/jcb.115.6.1561

Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells

PMCID: PMC2289208  PMID: 1661730

Abstract

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6- P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.

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Selected References

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  1. Bleekemolen J. E., Stein M., von Figura K., Slot J. W., Geuze H. J. The two mannose 6-phosphate receptors have almost identical subcellular distributions in U937 monocytes. Eur J Cell Biol. 1988 Dec;47(2):366–372. [PubMed] [Google Scholar]
  2. Bomsel M., Parton R., Kuznetsov S. A., Schroer T. A., Gruenberg J. Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytic vesicles from MDCK cells. Cell. 1990 Aug 24;62(4):719–731. doi: 10.1016/0092-8674(90)90117-w. [DOI] [PubMed] [Google Scholar]
  3. Brown W. J., Goodhouse J., Farquhar M. G. Mannose-6-phosphate receptors for lysosomal enzymes cycle between the Golgi complex and endosomes. J Cell Biol. 1986 Oct;103(4):1235–1247. doi: 10.1083/jcb.103.4.1235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cain C. C., Sipe D. M., Murphy R. F. Regulation of endocytic pH by the Na+,K+-ATPase in living cells. Proc Natl Acad Sci U S A. 1989 Jan;86(2):544–548. doi: 10.1073/pnas.86.2.544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Campbell C. H., Rome L. H. Coated vesicles from rat liver and calf brain contain lysosomal enzymes bound to mannose 6-phosphate receptors. J Biol Chem. 1983 Nov 10;258(21):13347–13352. [PubMed] [Google Scholar]
  6. Casciola-Rosen L. A., Hubbard A. L. Hydrolases in intracellular compartments of rat liver cells. Evidence for selective activation and/or delivery. J Biol Chem. 1991 Mar 5;266(7):4341–4347. [PubMed] [Google Scholar]
  7. Causin C., Waheed A., Braulke T., Junghans U., Maly P., Humbel R. E., von Figura K. Mannose 6-phosphate/insulin-like growth factor II-binding proteins in human serum and urine. Their relation to the mannose 6-phosphate/insulin-like growth factor II receptor. Biochem J. 1988 Jun 15;252(3):795–799. doi: 10.1042/bj2520795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chao H. H., Waheed A., Pohlmann R., Hille A., von Figura K. Mannose 6-phosphate receptor dependent secretion of lysosomal enzymes. EMBO J. 1990 Nov;9(11):3507–3513. doi: 10.1002/j.1460-2075.1990.tb07559.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dahms N. M., Lobel P., Kornfeld S. Mannose 6-phosphate receptors and lysosomal enzyme targeting. J Biol Chem. 1989 Jul 25;264(21):12115–12118. [PubMed] [Google Scholar]
  10. Diment S., Leech M. S., Stahl P. D. Cathepsin D is membrane-associated in macrophage endosomes. J Biol Chem. 1988 May 15;263(14):6901–6907. [PubMed] [Google Scholar]
  11. Diment S., Stahl P. Macrophage endosomes contain proteases which degrade endocytosed protein ligands. J Biol Chem. 1985 Dec 5;260(28):15311–15317. [PubMed] [Google Scholar]
  12. Doherty J. J., 2nd, Kay D. G., Lai W. H., Posner B. I., Bergeron J. J. Selective degradation of insulin within rat liver endosomes. J Cell Biol. 1990 Jan;110(1):35–42. doi: 10.1083/jcb.110.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Duncan J. R., Kornfeld S. Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus. J Cell Biol. 1988 Mar;106(3):617–628. doi: 10.1083/jcb.106.3.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Fischer H. D., Gonzalez-Noriega A., Sly W. S. Beta-glucuronidase binding to human fibroblast membrane receptors. J Biol Chem. 1980 Jun 10;255(11):5069–5074. [PubMed] [Google Scholar]
  16. Fuchs R., Schmid S., Mellman I. A possible role for Na+,K+-ATPase in regulating ATP-dependent endosome acidification. Proc Natl Acad Sci U S A. 1989 Jan;86(2):539–543. doi: 10.1073/pnas.86.2.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gabel C. A., Goldberg D. E., Kornfeld S. Identification and characterization of cells deficient in the mannose 6-phosphate receptor: evidence for an alternate pathway for lysosomal enzyme targeting. Proc Natl Acad Sci U S A. 1983 Feb;80(3):775–779. doi: 10.1073/pnas.80.3.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Geuze H. J., Slot J. W., Strous G. J., Hasilik A., von Figura K. Possible pathways for lysosomal enzyme delivery. J Cell Biol. 1985 Dec;101(6):2253–2262. doi: 10.1083/jcb.101.6.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Geuze H. J., Slot J. W., van der Ley P. A., Scheffer R. C. Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections. J Cell Biol. 1981 Jun;89(3):653–665. doi: 10.1083/jcb.89.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Geuze H. J., Stoorvogel W., Strous G. J., Slot J. W., Bleekemolen J. E., Mellman I. Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles. J Cell Biol. 1988 Dec;107(6 Pt 2):2491–2501. doi: 10.1083/jcb.107.6.2491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gieselmann V., Pohlmann R., Hasilik A., Von Figura K. Biosynthesis and transport of cathepsin D in cultured human fibroblasts. J Cell Biol. 1983 Jul;97(1):1–5. doi: 10.1083/jcb.97.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Goldberg D. E., Kornfeld S. The phosphorylation of beta-glucuronidase oligosaccharides in mouse P388D1 cells. J Biol Chem. 1981 Dec 25;256(24):13060–13067. [PubMed] [Google Scholar]
  23. Griffiths G., Back R., Marsh M. A quantitative analysis of the endocytic pathway in baby hamster kidney cells. J Cell Biol. 1989 Dec;109(6 Pt 1):2703–2720. doi: 10.1083/jcb.109.6.2703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Griffiths G., Hoflack B., Simons K., Mellman I., Kornfeld S. The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell. 1988 Feb 12;52(3):329–341. doi: 10.1016/s0092-8674(88)80026-6. [DOI] [PubMed] [Google Scholar]
  25. Griffiths G., Hollinshead R., Hemmings B. A., Nigg E. A. Ultrastructural localization of the regulatory (RII) subunit of cyclic AMP-dependent protein kinase to subcellular compartments active in endocytosis and recycling of membrane receptors. J Cell Sci. 1990 Aug;96(Pt 4):691–703. doi: 10.1242/jcs.96.4.691. [DOI] [PubMed] [Google Scholar]
  26. Griffiths G., Hoppeler H. Quantitation in immunocytochemistry: correlation of immunogold labeling to absolute number of membrane antigens. J Histochem Cytochem. 1986 Nov;34(11):1389–1398. doi: 10.1177/34.11.3534077. [DOI] [PubMed] [Google Scholar]
  27. Griffiths G., Matteoni R., Back R., Hoflack B. Characterization of the cation-independent mannose 6-phosphate receptor-enriched prelysosomal compartment in NRK cells. J Cell Sci. 1990 Mar;95(Pt 3):441–461. doi: 10.1242/jcs.95.3.441. [DOI] [PubMed] [Google Scholar]
  28. Gruenberg J., Griffiths G., Howell K. E. 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):1301–1316. doi: 10.1083/jcb.108.4.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Hasilik A., Neufeld E. F. Biosynthesis of lysosomal enzymes in fibroblasts. Synthesis as precursors of higher molecular weight. J Biol Chem. 1980 May 25;255(10):4937–4945. [PubMed] [Google Scholar]
  31. Hoflack B., Fujimoto K., Kornfeld S. The interaction of phosphorylated oligosaccharides and lysosomal enzymes with bovine liver cation-dependent mannose 6-phosphate receptor. J Biol Chem. 1987 Jan 5;262(1):123–129. [PubMed] [Google Scholar]
  32. Hubbard A. L. Endocytosis. Curr Opin Cell Biol. 1989 Aug;1(4):675–683. doi: 10.1016/0955-0674(89)90033-1. [DOI] [PubMed] [Google Scholar]
  33. Kornfeld S., Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. doi: 10.1146/annurev.cb.05.110189.002411. [DOI] [PubMed] [Google Scholar]
  34. Kyle J. W., Nolan C. M., Oshima A., Sly W. S. Expression of human cation-independent mannose 6-phosphate receptor cDNA in receptor-negative mouse P388D1 cells following gene transfer. J Biol Chem. 1988 Nov 5;263(31):16230–16235. [PubMed] [Google Scholar]
  35. Lemansky P., Hasilik A., von Figura K., Helmy S., Fishman J., Fine R. E., Kedersha N. L., Rome L. H. Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways. J Cell Biol. 1987 Jun;104(6):1743–1748. doi: 10.1083/jcb.104.6.1743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lobel P., Fujimoto K., Ye R. D., Griffiths G., Kornfeld S. Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis. Cell. 1989 Jun 2;57(5):787–796. doi: 10.1016/0092-8674(89)90793-9. [DOI] [PubMed] [Google Scholar]
  37. Mellman I., Galloway C. J. Selective labeling and quantitative analysis of internalized plasma membrane. Methods Enzymol. 1983;98:545–555. doi: 10.1016/0076-6879(83)98181-8. [DOI] [PubMed] [Google Scholar]
  38. Parton R. G., Prydz K., Bomsel M., Simons K., Griffiths G. Meeting of the apical and basolateral endocytic pathways of the Madin-Darby canine kidney cell in late endosomes. J Cell Biol. 1989 Dec;109(6 Pt 2):3259–3272. doi: 10.1083/jcb.109.6.3259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Sahagian G. G., Neufeld E. F. Biosynthesis and turnover of the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells. J Biol Chem. 1983 Jun 10;258(11):7121–7128. [PubMed] [Google Scholar]
  41. Sahagian G. G. The mannose 6-phosphate receptor: function, biosynthesis and translocation. Biol Cell. 1984;51(2):207–214. doi: 10.1111/j.1768-322x.1984.tb00300.x. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Slot J. W., Geuze H. J. A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol. 1985 Jul;38(1):87–93. [PubMed] [Google Scholar]
  44. Stein M., Zijderhand-Bleekemolen J. E., Geuze H., Hasilik A., von Figura K. Mr 46,000 mannose 6-phosphate specific receptor: its role in targeting of lysosomal enzymes. EMBO J. 1987 Sep;6(9):2677–2681. doi: 10.1002/j.1460-2075.1987.tb02559.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Steinman R. M., Cohn Z. A. The interaction of soluble horseradish peroxidase with mouse peritoneal macrophages in vitro. J Cell Biol. 1972 Oct;55(1):186–204. doi: 10.1083/jcb.55.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tong P. Y., Kornfeld S. Ligand interactions of the cation-dependent mannose 6-phosphate receptor. Comparison with the cation-independent mannose 6-phosphate receptor. J Biol Chem. 1989 May 15;264(14):7970–7975. [PubMed] [Google Scholar]
  47. Watts C. In situ 125I-labelling of endosome proteins with lactoperoxidase conjugates. EMBO J. 1984 Sep;3(9):1965–1970. doi: 10.1002/j.1460-2075.1984.tb02077.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Willingham M. C., Pastan I. H., Sahagian G. G. Ultrastructural immunocytochemical localization of the phosphomannosyl receptor in Chinese hamster ovary (CHO) cells. J Histochem Cytochem. 1983 Jan;31(1):1–11. doi: 10.1177/31.1.6300218. [DOI] [PubMed] [Google Scholar]
  49. Yewdell J. W., Bennink J. R. The binary logic of antigen processing and presentation to T cells. Cell. 1990 Jul 27;62(2):203–206. doi: 10.1016/0092-8674(90)90356-j. [DOI] [PubMed] [Google Scholar]
  50. von Figura K., Gieselmann V., Hasilik A. Antibody to mannose 6-phosphate specific receptor induces receptor deficiency in human fibroblasts. EMBO J. 1984 Jun;3(6):1281–1286. doi: 10.1002/j.1460-2075.1984.tb01963.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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