Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Feb 1;100(2):535–544. doi: 10.1083/jcb.100.2.535

Processing and lysosomal localization of a glycoprotein whose secretion is transformation stimulated

PMCID: PMC2113454  PMID: 3968177

Abstract

The major excreted protein (MEP) of transformed mouse fibroblasts is a mannose 6-phosphate-containing glycoprotein whose synthesis and secretion are increased in malignantly transformed 3T3 cells and whose synthesis is increased by treatment of 3T3 cells with tumor promoters or growth factors. When pulse-labeled extracts from Kirsten virus- transformed NIH 3T3 (KNIH) cells were immunoprecipitated using an antibody against secreted MEP, one cellular protein was immunoprecipitated that had the same molecular weight and tryptic peptide map as the secreted protein. Pulse-chase labeling experiments showed that 50-60% of this 39,000-mol-wt form was secreted in transformed cells. Of the 40-50% remaining, approximately 5% was processed into two lower molecular weight forms (29,000 and 20,000) which are sequestered within the cell. Similar processing of these proteins was observed in the nontransformed parent NIH 3T3 (NIH) cells. However, in NIH cells, much less of the synthesized MEP was secreted. Measurements of steady-state levels of these three forms of cellular MEP by Western blot immunolocalization revealed approximately fourfold more MEP in KNIH cells than in NIH cells as well as differences in the relative distribution of MEP forms in transformed and nontransformed cells. Subcellular fractionation of KNIH cells on a Percoll gradient demonstrated a distribution of total MEP similar to that of several lysosomal enzymes. The light lysosomal/Golgi peak from these gradients contained both the precursor 39,000-mol-wt form of MEP and the 20,000- mol-wt form, whereas the heavy lysosomal peak was enriched in the 20,000-mol-wt form. The distribution of MEP forms was found to be similar in NIH cells except that the 29,000-mol-wt form was also seen to be enriched in the heavy lysosomal peak. This biochemical localization of MEP was confirmed by immunolocalization with light and electron microscopy. These data support the hypothesis that MEP is a lysosomal protein that is secreted by transformed cells.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Baumbach G. A., Saunders P. T., Bazer F. W., Roberts R. M. Uteroferrin has N-asparagine-linked high-mannose-type oligosaccharides that contain mannose 6-phosphate. Proc Natl Acad Sci U S A. 1984 May;81(10):2985–2989. doi: 10.1073/pnas.81.10.2985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  3. Bonner W. M., Stedman J. D. Efficient fluorography of 3H and 14C on thin layers. Anal Biochem. 1978 Aug 15;89(1):247–256. doi: 10.1016/0003-2697(78)90747-9. [DOI] [PubMed] [Google Scholar]
  4. Bowers W. E., de Duve C. Lysosomes in lymphoid tissue. II. Intracellular distribution of acid hydrolases. J Cell Biol. 1967 Feb;32(2):339–348. doi: 10.1083/jcb.32.2.339. [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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Cabral F., Abraham I., Gottesman M. M. Revertants of a Chinese hamster ovary cell mutant with an altered beta-tubulin: evidence that the altered tubulin confers both colcemid resistance and temperature sensitivity on the cell. Mol Cell Biol. 1982 Jun;2(6):720–729. doi: 10.1128/mcb.2.6.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cabral F., Gottesman M. M., Yuspa S. H. Induction of specific protein synthesis by phorbol esters in mouse epidermal cell culture. Cancer Res. 1981 Jun;41(6):2025–2031. [PubMed] [Google Scholar]
  8. Cabral F., Schatz G. High resolution one- and two- dimensional electrophoretic analysis of mitochondrial membrane polypeptides. Methods Enzymol. 1979;56:602–613. doi: 10.1016/0076-6879(79)56057-1. [DOI] [PubMed] [Google Scholar]
  9. Cabral F., Willingham M. C., Gottesman M. M. Ultrastructural localization to 10 nm filaments of an insoluble 58K protein in cultured fibroblasts. J Histochem Cytochem. 1980 Jul;28(7):653–662. doi: 10.1177/28.7.7391554. [DOI] [PubMed] [Google Scholar]
  10. Fleischer B., Fleischer S., Ozawa H. Isolation and characterization of Golgi membranes from bovine liver. J Cell Biol. 1969 Oct;43(1):59–79. doi: 10.1083/jcb.43.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Gonzalez-Noriega A., Grubb J. H., Talkad V., Sly W. S. Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling. J Cell Biol. 1980 Jun;85(3):839–852. doi: 10.1083/jcb.85.3.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gottesman M. M., Cabral F. Purification and characterization of a transformation-dependent protein secreted by cultured murine fibroblasts. Biochemistry. 1981 Mar 17;20(6):1659–1665. doi: 10.1021/bi00509a039. [DOI] [PubMed] [Google Scholar]
  14. Gottesman M. M., Sobel M. E. Tumor promoters and Kirsten sarcoma virus increase synthesis of a secreted glycoprotein by regulating levels of translatable mRNA. Cell. 1980 Feb;19(2):449–455. doi: 10.1016/0092-8674(80)90519-x. [DOI] [PubMed] [Google Scholar]
  15. Gottesman M. M. Transformation-dependent secretion of a low molecular weight protein by murine fibroblasts. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2767–2771. doi: 10.1073/pnas.75.6.2767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Green H., Todaro G. J., Goldberg B. Collagen synthesis in fibroblasts transformed by oncogenic viruses. Nature. 1966 Feb 26;209(5026):916–917. doi: 10.1038/209916a0. [DOI] [PubMed] [Google Scholar]
  17. Hasilik A., Waheed A., von Figura K. Enzymatic phosphorylation of lysosomal enzymes in the presence of UDP-N-acetylglucosamine. Absence of the activity in I-cell fibroblasts. Biochem Biophys Res Commun. 1981 Feb 12;98(3):761–767. doi: 10.1016/0006-291x(81)91177-3. [DOI] [PubMed] [Google Scholar]
  18. Horvat A., Touster O. On the lysosomal occurrence and the properties of the neuraminidase of rat liver and of Ehrlich ascites tumor cells. J Biol Chem. 1968 Aug 25;243(16):4380–4390. [PubMed] [Google Scholar]
  19. 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]
  20. McDonald J. K., Reilly T. J., Zeitman B. B., Ellis S. Dipeptidyl arylamidase II of the pituitary. Properties of lysylalanyl-beta-naphthylamide hydrolysis: inhibition by cations, distribution in tissues, and subcellular localization. J Biol Chem. 1968 Apr 25;243(8):2028–2037. [PubMed] [Google Scholar]
  21. Nilsen-Hamilton M., Hamilton R. T., Allen W. R., Massoglia S. L. Stimulation of the release of two glycoproteins from mouse 3T3 cells by growth factors and by agents that increase intralysosomal pH. Biochem Biophys Res Commun. 1981 Jul 30;101(2):411–417. doi: 10.1016/0006-291x(81)91275-4. [DOI] [PubMed] [Google Scholar]
  22. Owada M., Neufeld E. F. Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? Evidence from I-cell disease. Biochem Biophys Res Commun. 1982 Apr 14;105(3):814–820. doi: 10.1016/0006-291x(82)91042-7. [DOI] [PubMed] [Google Scholar]
  23. Pastan I., Willingham M. Cellular transformation and the 'morphologic phenotype' of transformed cells. Nature. 1978 Aug 17;274(5672):645–650. doi: 10.1038/274645a0. [DOI] [PubMed] [Google Scholar]
  24. Reitman M. L., Varki A., Kornfeld S. Fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy are deficient in uridine 5'-diphosphate-N-acetylglucosamine: glycoprotein N-acetylglucosaminylphosphotransferase activity. J Clin Invest. 1981 May;67(5):1574–1579. doi: 10.1172/JCI110189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Robbins A. R., Myerowitz R. The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors. J Biol Chem. 1981 Oct 25;256(20):10623–10627. [PubMed] [Google Scholar]
  26. Robbins A. R., Peng S. S., Marshall J. L. Mutant Chinese hamster ovary cells pleiotropically defective in receptor-mediated endocytosis. J Cell Biol. 1983 Apr;96(4):1064–1071. doi: 10.1083/jcb.96.4.1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Sahagian G. G., Gottesman M. M. The predominant secreted protein of transformed murine fibroblasts carries the lysosomal mannose 6-phosphate recognition marker. J Biol Chem. 1982 Sep 25;257(18):11145–11150. [PubMed] [Google Scholar]
  29. Shih T. Y., Williams D. R., Weeks M. O., Maryak J. M., Vass W. C., Scolnick E. M. Comparison of the genomic organization of Kirsten and Harvey sarcoma viruses. J Virol. 1978 Jul;27(1):45–55. doi: 10.1128/jvi.27.1.45-55.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sloane B. F., Dunn J. R., Honn K. V. Lysosomal cathepsin B: correlation with metastatic potential. Science. 1981 Jun 5;212(4499):1151–1153. doi: 10.1126/science.7233209. [DOI] [PubMed] [Google Scholar]
  31. Snellman O. Cathepsin B, the lysosomal thiol proteinase of calf liver. Biochem J. 1969 Oct;114(4):673–678. doi: 10.1042/bj1140673b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Unkeless J. C., Tobia A., Ossowski L., Quigley J. P., Rifkin D. B., Reich E. An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. I. Chick embryo fibroblast cultures transformed by avian RNA tumor viruses. J Exp Med. 1973 Jan 1;137(1):85–111. doi: 10.1084/jem.137.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vaes G. Hyaluronidase activity in lysosomes of bone tissue. Biochem J. 1967 Jun;103(3):802–804. doi: 10.1042/bj1030802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Waheed A., Pohlmann R., Hasilik A., von Figura K., van Elsen A., Leroy J. G. Deficiency of UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase in organs of I-cell patients. Biochem Biophys Res Commun. 1982 Apr 14;105(3):1052–1058. doi: 10.1016/0006-291x(82)91076-2. [DOI] [PubMed] [Google Scholar]
  36. Waite M., Scherphof G. L., Boshouwers F. M., van Deenen L. L. Differentiation of phospholipases A in mitochondria and lysosomes of rat liver. J Lipid Res. 1969 Jul;10(4):411–420. [PubMed] [Google Scholar]
  37. Willingham M. C. Electron microscopic immunocytochemical localization of intracellular antigens in cultured cells: the EGS and ferritin bridge procedures. Histochem J. 1980 Jul;12(4):419–434. doi: 10.1007/BF01011958. [DOI] [PubMed] [Google Scholar]
  38. Wynn C. H. Solubilization of insoluble collagens by rat liver lysosomes. Nature. 1967 Sep 9;215(5106):1191–1192. doi: 10.1038/2151191a0. [DOI] [PubMed] [Google Scholar]
  39. Yamada K. M., Olden K. Fibronectins--adhesive glycoproteins of cell surface and blood. Nature. 1978 Sep 21;275(5677):179–184. doi: 10.1038/275179a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES