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. 1989 Jun 1;108(6):2149–2162. doi: 10.1083/jcb.108.6.2149

Isolation and characterization of membranes from bovine liver which are highly enriched in mannose 6-phosphate receptors

PMCID: PMC2115605  PMID: 2544603

Abstract

We have developed a method for the isolation of the subcellular organelles from bovine liver which are enriched in the cation- independent mannose 6-phosphate receptor (CI-MPR) and the cation- dependent mannose 6-phosphate receptor (CD-MPR). The purification scheme consists of sedimentation of a postnuclear supernatant fraction on a sucrose gradient followed by immunoisolation using specific anti- peptide antibodies conjugated to magnetic polystyrene beads. Antibodies that recognize the cytoplasmic domain of either the CI-MPR or the CD- MPR routinely give membrane preparations that are approximately 50-fold enriched in each of the respective receptors, as determined by quantitative Western blotting. The immunoisolated membranes are also enriched in the other MPR, as well as in the asialoglycoprotein receptor. They contain significantly lower levels of enzyme activities representative of the plasma membrane (5' nucleotidase) or the Golgi complex (galactosyltransferase and sialyltransferase). There is little or no enrichment for either the lysosomal enzymes beta-hexosaminidase and tartrate-resistant acid phosphatase, or the mitochondrial enzyme succinate-tetrazolium reductase. These data, together with electron microscopy of the immunoisolated material, suggest that the bulk of MPR- containing membranes we have isolated from bovine liver correspond to endosomes. Analysis by SDS-PAGE indicates that several proteins, including two with apparent molecular weights of 170 K and 400 K, are significantly enriched in the purified fractions and may represent potential markers for MPR-containing endosomes.

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Selected References

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  1. Bartles J. R., Braiterman L. T., Hubbard A. L. Endogenous and exogenous domain markers of the rat hepatocyte plasma membrane. J Cell Biol. 1985 Apr;100(4):1126–1138. doi: 10.1083/jcb.100.4.1126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berger E. G., Thurnher M., Müller U. Galactosyltransferase and sialyltransferase are located in different subcellular compartments in HeLa cells. Exp Cell Res. 1987 Nov;173(1):267–273. doi: 10.1016/0014-4827(87)90352-1. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bretz R., Stäubli W. Detergent influence on rat-liver galactosyltransferase activities towards different acceptors. Eur J Biochem. 1977 Jul 1;77(1):181–192. doi: 10.1111/j.1432-1033.1977.tb11656.x. [DOI] [PubMed] [Google Scholar]
  5. Briles E. B., Li E., Kornfeld S. Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose. J Biol Chem. 1977 Feb 10;252(3):1107–1116. [PubMed] [Google Scholar]
  6. Brown W. J., Farquhar M. G. The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis Golgi cisternae. Cell. 1984 Feb;36(2):295–307. doi: 10.1016/0092-8674(84)90223-x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Dahms N. M., Lobel P., Breitmeyer J., Chirgwin J. M., Kornfeld S. 46 kd mannose 6-phosphate receptor: cloning, expression, and homology to the 215 kd mannose 6-phosphate receptor. Cell. 1987 Jul 17;50(2):181–192. doi: 10.1016/0092-8674(87)90214-5. [DOI] [PubMed] [Google Scholar]
  10. Devaney E., Howell K. E. Immuno-isolation of a plasma membrane fraction from the Fao cell. EMBO J. 1985 Dec 1;4(12):3123–3130. doi: 10.1002/j.1460-2075.1985.tb04054.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Fischer H. D., Gonzalez-Noriega A., Sly W. S., Morré D. J. Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes. J Biol Chem. 1980 Oct 25;255(20):9608–9615. [PubMed] [Google Scholar]
  14. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Geuze H. J., Slot J. W., Strous G. J., Hasilik A., Von Figura K. Ultrastructural localization of the mannose 6-phosphate receptor in rat liver. J Cell Biol. 1984 Jun;98(6):2047–2054. doi: 10.1083/jcb.98.6.2047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  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., 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. 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]
  21. Gieselmann V., Hasilik A., von Figura K. Tartrate-inhibitable acid phosphatase. Purification from placenta, characterization and subcellular distribution in fibroblasts. Hoppe Seylers Z Physiol Chem. 1984 Jun;365(6):651–660. doi: 10.1515/bchm2.1984.365.1.651. [DOI] [PubMed] [Google Scholar]
  22. Gottlieb C., Baenziger J., Kornfeld S. Deficient uridine diphosphate-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activity in a clone of Chinese hamster ovary cells with altered surface glycoproteins. J Biol Chem. 1975 May 10;250(9):3303–3309. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Griffiths G., McDowall A., Back R., Dubochet J. On the preparation of cryosections for immunocytochemistry. J Ultrastruct Res. 1984 Oct;89(1):65–78. doi: 10.1016/s0022-5320(84)80024-6. [DOI] [PubMed] [Google Scholar]
  26. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  27. Griffiths G., Warren G., Quinn P., Mathieu-Costello O., Hoppeler H. Density of newly synthesized plasma membrane proteins in intracellular membranes. I. Stereological studies. J Cell Biol. 1984 Jun;98(6):2133–2141. doi: 10.1083/jcb.98.6.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Gruenberg J., Howell K. E. Immuno-isolation of vesicles using antigenic sites either located on the cytoplasmic or the exoplasmic domain of an implanted viral protein. A quantitative analysis. Eur J Cell Biol. 1985 Sep;38(2):312–321. [PubMed] [Google Scholar]
  29. Hand A. R. Cytochemical differentiation of the Golgi apparatus from GERL. J Histochem Cytochem. 1980 Jan;28(1):82–86. doi: 10.1177/28.1.7351475. [DOI] [PubMed] [Google Scholar]
  30. Hickman S., Neufeld E. F. A hypothesis for I-cell disease: defective hydrolases that do not enter lysosomes. Biochem Biophys Res Commun. 1972 Nov 15;49(4):992–999. doi: 10.1016/0006-291x(72)90310-5. [DOI] [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. Hoflack B., Kornfeld S. Lysosomal enzyme binding to mouse P388D1 macrophage membranes lacking the 215-kDa mannose 6-phosphate receptor: evidence for the existence of a second mannose 6-phosphate receptor. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4428–4432. doi: 10.1073/pnas.82.13.4428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hoflack B., Kornfeld S. Purification and characterization of a cation-dependent mannose 6-phosphate receptor from murine P388D1 macrophages and bovine liver. J Biol Chem. 1985 Oct 5;260(22):12008–12014. [PubMed] [Google Scholar]
  34. 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]
  35. KILSHEIMER G. S., AXELROD B. Phylogenetic distribution of acid phosphatase inhibited by (+)-tartrate. Nature. 1958 Dec 20;182(4651):1733–1734. doi: 10.1038/1821733a0. [DOI] [PubMed] [Google Scholar]
  36. Kornfeld S. Trafficking of lysosomal enzymes. FASEB J. 1987 Dec;1(6):462–468. doi: 10.1096/fasebj.1.6.3315809. [DOI] [PubMed] [Google Scholar]
  37. Lobel P., Dahms N. M., Kornfeld S. Cloning and sequence analysis of the cation-independent mannose 6-phosphate receptor. J Biol Chem. 1988 Feb 15;263(5):2563–2570. [PubMed] [Google Scholar]
  38. Marsh M., Schmid S., Kern H., Harms E., Male P., Mellman I., Helenius A. Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis. J Cell Biol. 1987 Apr;104(4):875–886. doi: 10.1083/jcb.104.4.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  41. Oshima A., Nolan C. M., Kyle J. W., Grubb J. H., Sly W. S. The human cation-independent mannose 6-phosphate receptor. Cloning and sequence of the full-length cDNA and expression of functional receptor in COS cells. J Biol Chem. 1988 Feb 15;263(5):2553–2562. [PubMed] [Google Scholar]
  42. PENNINGTON R. J. Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J. 1961 Sep;80:649–654. doi: 10.1042/bj0800649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Pohlmann R., Nagel G., Schmidt B., Stein M., Lorkowski G., Krentler C., Cully J., Meyer H. E., Grzeschik K. H., Mersmann G. Cloning of a cDNA encoding the human cation-dependent mannose 6-phosphate-specific receptor. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5575–5579. doi: 10.1073/pnas.84.16.5575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Roman L. M., Hubbard A. L. A domain-specific marker for the hepatocyte plasma membrane. III. Isolation of bile canalicular membrane by immunoadsorption. J Cell Biol. 1984 Apr;98(4):1497–1504. doi: 10.1083/jcb.98.4.1497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Roth J., Berger E. G. Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae. J Cell Biol. 1982 Apr;93(1):223–229. doi: 10.1083/jcb.93.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Roth J., Taatjes D. J., Lucocq J. M., Weinstein J., Paulson J. C. Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation. Cell. 1985 Nov;43(1):287–295. doi: 10.1016/0092-8674(85)90034-0. [DOI] [PubMed] [Google Scholar]
  48. Sahagian G. G., Distler J., Jourdian G. W. Characterization of a membrane-associated receptor from bovine liver that binds phosphomannosyl residues of bovine testicular beta-galactosidase. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4289–4293. doi: 10.1073/pnas.78.7.4289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Sahagian G. G., Steer C. J. Transmembrane orientation of the mannose 6-phosphate receptor in isolated clathrin-coated vesicles. J Biol Chem. 1985 Aug 15;260(17):9838–9842. [PubMed] [Google Scholar]
  51. Stein M., Braulke T., Krentler C., Hasilik A., von Figura K. 46-kDa mannose 6-phosphate-specific receptor: biosynthesis, processing, subcellular location and topology. Biol Chem Hoppe Seyler. 1987 Aug;368(8):937–947. doi: 10.1515/bchm3.1987.368.2.937. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Waheed A., Gottschalk S., Hille A., Krentler C., Pohlmann R., Braulke T., Hauser H., Geuze H., von Figura K. Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells. EMBO J. 1988 Aug;7(8):2351–2358. doi: 10.1002/j.1460-2075.1988.tb03079.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wall D. A., Hubbard A. L. Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments. J Cell Biol. 1985 Dec;101(6):2104–2112. doi: 10.1083/jcb.101.6.2104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. 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]
  57. Zijderhand-Bleekemolen J. E., Schwartz A. L., Slot J. W., Strous G. J., Geuze H. J. Ligand- and weak base-induced redistribution of asialoglycoprotein receptors in hepatoma cells. J Cell Biol. 1987 Jun;104(6):1647–1654. doi: 10.1083/jcb.104.6.1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. von Figura K., Hasilik A. Lysosomal enzymes and their receptors. Annu Rev Biochem. 1986;55:167–193. doi: 10.1146/annurev.bi.55.070186.001123. [DOI] [PubMed] [Google Scholar]

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