Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 May;11(5):2362–2374. doi: 10.1128/mcb.11.5.2362

Anchoring and degradation of glycolipid-anchored membrane proteins by L929 versus by LM-TK- mouse fibroblasts: implications for anchor biosynthesis.

N Singh 1, D Singleton 1, A M Tartakoff 1
PMCID: PMC359988  PMID: 1826759

Abstract

Although many cells anchor surface proteins via moieties that are sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC), the anchor moieties of surface proteins of mouse L929 cells resist PI-PLC. By constructing stable hybrids between L929 and lymphoma cells that express glycolipid-anchored proteins in a PI-PLC-sensitive form, we show that PI-PLC resistance behaves as a recessive trait. Since putative mannolipid precursors of the lipid anchors bear alkali-labile substituents which make them resist PI-PLC, these observations are most simply interpreted by postulating that L929 lacks a critical anchor deacylase. Unlike the L929 cell line, two of its descendants, the LM cell line and its thymidine kinase-negative variant (LM-TK-), do not express glycolipid-anchored proteins on their surface. Moreover, unlike L929 cells, LM-TK- cells rapidly inactivate at least one lipid-anchored enzyme in a compartment sensitive to acidotropic amines and leupeptin. By fusion of LM-TK- cells to mouse Thy-1- lymphoma mutants and monitoring of surface expression of lipid-anchored proteins, we assign LM-TK- to lymphoma mutant complementation group H. This genetic assignment is matched by analysis of mannolipids of L929, LM-TK-, wild-type, and class H lymphoma mutant cells: striking similarities are seen between the two wild-type cells by contrast to the mutants. Since the differences pertain to lipids which have properties consistent with their being anchor precursors, we suggest that LM-TK- has a lesion in the synthesis of anchor precursor mannolipids.

Full text

PDF
2369

Images in this article

2365Image
on p.2365
2367Image
on p.2367
2368Image
on p.2368
2370Image
on p.2370
2371Image
on p.2371
2372Image
on p.2372
2372Image
on p.2372

Selected References

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

  1. Andreason G. L., Evans G. A. Optimization of electroporation for transfection of mammalian cell lines. Anal Biochem. 1989 Aug 1;180(2):269–275. doi: 10.1016/0003-2697(89)90429-6. [DOI] [PubMed] [Google Scholar]
  2. Bangs J. D., Hereld D., Krakow J. L., Hart G. W., Englund P. T. Rapid processing of the carboxyl terminus of a trypanosome variant surface glycoprotein. Proc Natl Acad Sci U S A. 1985 May;82(10):3207–3211. doi: 10.1073/pnas.82.10.3207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bender M. A., Palmer T. D., Gelinas R. E., Miller A. D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol. 1987 May;61(5):1639–1646. doi: 10.1128/jvi.61.5.1639-1646.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berger J., Howard A. D., Brink L., Gerber L., Hauber J., Cullen B. R., Udenfriend S. COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein. J Biol Chem. 1988 Jul 15;263(20):10016–10021. [PubMed] [Google Scholar]
  5. Berger J., Howard A. D., Gerber L., Cullen B. R., Udenfriend S. Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4885–4889. doi: 10.1073/pnas.84.14.4885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bonifacino J. S., Suzuki C. K., Klausner R. D. A peptide sequence confers retention and rapid degradation in the endoplasmic reticulum. Science. 1990 Jan 5;247(4938):79–82. doi: 10.1126/science.2294595. [DOI] [PubMed] [Google Scholar]
  7. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  8. Conzelmann A., Riezman H., Desponds C., Bron C. A major 125-kd membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid. EMBO J. 1988 Jul;7(7):2233–2240. doi: 10.1002/j.1460-2075.1988.tb03063.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Conzelmann A., Spiazzi A., Bron C., Hyman R. No glycolipid anchors are added to Thy-1 glycoprotein in Thy-1-negative mutant thymoma cells of four different complementation classes. Mol Cell Biol. 1988 Feb;8(2):674–678. doi: 10.1128/mcb.8.2.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Conzelmann A., Spiazzi A., Hyman R., Bron C. Anchoring of membrane proteins via phosphatidylinositol is deficient in two classes of Thy-1 negative mutant lymphoma cells. EMBO J. 1986 Dec 1;5(12):3291–3296. doi: 10.1002/j.1460-2075.1986.tb04642.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cross G. A. Eukaryotic protein modification and membrane attachment via phosphatidylinositol. Cell. 1987 Jan 30;48(2):179–181. doi: 10.1016/0092-8674(87)90419-3. [DOI] [PubMed] [Google Scholar]
  12. Edelman G. M., Murray B. A., Mege R. M., Cunningham B. A., Gallin W. J. Cellular expression of liver and neural cell adhesion molecules after transfection with their cDNAs results in specific cell-cell binding. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8502–8506. doi: 10.1073/pnas.84.23.8502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Endo T., Ohbayashi H., Hayashi Y., Ikehara Y., Kochibe N., Kobata A. Structural study on the carbohydrate moiety of human placental alkaline phosphatase. J Biochem. 1988 Jan;103(1):182–187. doi: 10.1093/oxfordjournals.jbchem.a122228. [DOI] [PubMed] [Google Scholar]
  14. Evans G. A., Ingraham H. A., Lewis K., Cunningham K., Seki T., Moriuchi T., Chang H. C., Silver J., Hyman R. Expression of the Thy-1 glycoprotein gene by DNA-mediated gene transfer. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5532–5536. doi: 10.1073/pnas.81.17.5532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fatemi S. H., Haas R., Jentoft N., Rosenberry T. L., Tartakoff A. M. The glycophospholipid anchor of Thy-1. Biosynthetic labeling experiments with wild-type and class E Thy-1 negative lymphomas. J Biol Chem. 1987 Apr 5;262(10):4728–4732. [PubMed] [Google Scholar]
  16. Fatemi S. H., Tartakoff A. M. Hydrophilic anchor-deficient Thy-1 is secreted by a class E mutant T lymphoma. Cell. 1986 Aug 29;46(5):653–657. doi: 10.1016/0092-8674(86)90340-5. [DOI] [PubMed] [Google Scholar]
  17. Fatemi S. H., Tartakoff A. M. The phenotype of five classes of T lymphoma mutants. Defective glycophospholipid anchoring, rapid degradation, and secretion of Thy-1 glycoprotein. J Biol Chem. 1988 Jan 25;263(3):1288–1294. [PubMed] [Google Scholar]
  18. Ferguson M. A., Duszenko M., Lamont G. S., Overath P., Cross G. A. Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-glycosylation and addition of a phosphatidylinositol membrane anchor. J Biol Chem. 1986 Jan 5;261(1):356–362. [PubMed] [Google Scholar]
  19. Ferguson M. A., Williams A. F. Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Annu Rev Biochem. 1988;57:285–320. doi: 10.1146/annurev.bi.57.070188.001441. [DOI] [PubMed] [Google Scholar]
  20. Gaulton G. N., Kelly K. L., Pawlowski J., Mato J. M., Jarett L. Regulation and function of an insulin-sensitive glycosyl-phosphatidylinositol during T lymphocyte activation. Cell. 1988 Jun 17;53(6):963–970. doi: 10.1016/s0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
  21. Gum J. R., Jr, Raetz C. R. Biochemical and immunological characterization of mutant L-M cells with altered levels of dibutyryl cyclic AMP-inducible alkaline phosphatase. Mol Cell Biol. 1985 May;5(5):1184–1187. doi: 10.1128/mcb.5.5.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gum J. R., Jr, Raetz C. R. Dibutyryl cAMP-inducible alkaline phosphatase in animal cell plasma membranes: fluorescence detection of mutant clones on polyester cloth. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3918–3922. doi: 10.1073/pnas.80.13.3918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gunter K. C., Kroczek R. A., Shevach E. M., Germain R. N. Functional expression of the murine Thy-1.2 gene in transfected human T cells. J Exp Med. 1986 Feb 1;163(2):285–300. doi: 10.1084/jem.163.2.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Henthorn P., Zervos P., Raducha M., Harris H., Kadesch T. Expression of a human placental alkaline phosphatase gene in transfected cells: use as a reporter for studies of gene expression. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6342–6346. doi: 10.1073/pnas.85.17.6342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hyman R., Kelleher R. J., Jr Absence of Thy-1 antigen in L-cell X mouse lymphoma hybrids. Somatic Cell Genet. 1975 Oct;1(4):335–343. doi: 10.1007/BF01538665. [DOI] [PubMed] [Google Scholar]
  26. Hyman R. Somatic genetic analysis of the expression of cell surface molecules. Trends Genet. 1988 Jan;4(1):5–8. doi: 10.1016/0168-9525(88)90120-5. [DOI] [PubMed] [Google Scholar]
  27. Isobe K., Fortunato A., Giguere V., Grosveld F., Mitchison N. A. Anti-Thy-1 antibody responses evoked by Thy-1 antigen expressed in transfected mouse mastocytoma cells and rat fibroblast. Immunology. 1985 Nov;56(3):505–512. [PMC free article] [PubMed] [Google Scholar]
  28. KIT S., DUBBS D. R., PIEKARSKI L. J., HSU T. C. DELETION OF THYMIDINE KINASE ACTIVITY FROM L CELLS RESISTANT TO BROMODEOXYURIDINE. Exp Cell Res. 1963 Aug;31:297–312. doi: 10.1016/0014-4827(63)90007-7. [DOI] [PubMed] [Google Scholar]
  29. Kimura S., Tada N., Liu-Lam Y., Hämmerling U. Studies of the mouse Ly-6 alloantigen system. II. Complexities of the Ly-6 region. Immunogenetics. 1984;20(1):47–56. doi: 10.1007/BF00373446. [DOI] [PubMed] [Google Scholar]
  30. Krakow J. L., Hereld D., Bangs J. D., Hart G. W., Englund P. T. Identification of a glycolipid precursor of the Trypanosoma brucei variant surface glycoprotein. J Biol Chem. 1986 Sep 15;261(26):12147–12153. [PubMed] [Google Scholar]
  31. LeClair K. P., Palfree R. G., Flood P. M., Hammerling U., Bothwell A. Isolation of a murine Ly-6 cDNA reveals a new multigene family. EMBO J. 1986 Dec 1;5(12):3227–3234. doi: 10.1002/j.1460-2075.1986.tb04633.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lemansky P., Fatemi S. H., Gorican B., Meyale S., Rossero R., Tartakoff A. M. Dynamics and longevity of the glycolipid-anchored membrane protein, Thy-1. J Cell Biol. 1990 May;110(5):1525–1531. doi: 10.1083/jcb.110.5.1525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lippincott-Schwartz J., Bonifacino J. S., Yuan L. C., Klausner R. D. Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins. Cell. 1988 Jul 15;54(2):209–220. doi: 10.1016/0092-8674(88)90553-3. [DOI] [PubMed] [Google Scholar]
  34. Low M. G., Prasad A. R. A phospholipase D specific for the phosphatidylinositol anchor of cell-surface proteins is abundant in plasma. Proc Natl Acad Sci U S A. 1988 Feb;85(4):980–984. doi: 10.1073/pnas.85.4.980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. MUNYON W. H., MERCHANT D. J. The relation between glucose utilization, lactic acid production and utilization and the growth cycle of L strain fibroblasts. Exp Cell Res. 1959 Jun;17(3):490–498. doi: 10.1016/0014-4827(59)90069-2. [DOI] [PubMed] [Google Scholar]
  36. Masterson W. J., Doering T. L., Hart G. W., Englund P. T. A novel pathway for glycan assembly: biosynthesis of the glycosyl-phosphatidylinositol anchor of the trypanosome variant surface glycoprotein. Cell. 1989 Mar 10;56(5):793–800. doi: 10.1016/0092-8674(89)90684-3. [DOI] [PubMed] [Google Scholar]
  37. Mato J. M., Kelly K. L., Abler A., Jarett L. Identification of a novel insulin-sensitive glycophospholipid from H35 hepatoma cells. J Biol Chem. 1987 Feb 15;262(5):2131–2137. [PubMed] [Google Scholar]
  38. Micanovic R., Bailey C. A., Brink L., Gerber L., Pan Y. C., Hulmes J. D., Udenfriend S. Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1398–1402. doi: 10.1073/pnas.85.5.1398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Micanovic R., Gerber L. D., Berger J., Kodukula K., Udenfriend S. Selectivity of the cleavage/attachment site of phosphatidylinositol-glycan-anchored membrane proteins determined by site-specific mutagenesis at Asp-484 of placental alkaline phosphatase. Proc Natl Acad Sci U S A. 1990 Jan;87(1):157–161. doi: 10.1073/pnas.87.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nilsson T., Jackson M., Peterson P. A. Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum. Cell. 1989 Aug 25;58(4):707–718. doi: 10.1016/0092-8674(89)90105-0. [DOI] [PubMed] [Google Scholar]
  41. Ogata S., Hayashi Y., Takami N., Ikehara Y. Chemical characterization of the membrane-anchoring domain of human placental alkaline phosphatase. J Biol Chem. 1988 Jul 25;263(21):10489–10494. [PubMed] [Google Scholar]
  42. Rettig W. J., Nishimura H., Yenamandra A. K., Seki T., Obata F., Beresford H. R., Old L. J., Silver J. Differential expression of the human Thy-1 gene in rodent-human somatic cell hybrids [corrected]. J Immunol. 1987 Jun 15;138(12):4484–4489. [PubMed] [Google Scholar]
  43. Roberts W. L., Myher J. J., Kuksis A., Low M. G., Rosenberry T. L. Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase C. J Biol Chem. 1988 Dec 15;263(35):18766–18775. [PubMed] [Google Scholar]
  44. Roberts W. L., Santikarn S., Reinhold V. N., Rosenberry T. L. Structural characterization of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase by fast atom bombardment mass spectrometry. J Biol Chem. 1988 Dec 15;263(35):18776–18784. [PubMed] [Google Scholar]
  45. Rosenberry T. L., Krall J. A., Dever T. E., Haas R., Louvard D., Merrick W. C. Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1 alpha reveals a new post-translational protein modification. J Biol Chem. 1989 May 5;264(13):7096–7099. [PubMed] [Google Scholar]
  46. SANFORD K. K., EARLE W. R., LIKELY G. D. The growth in vitro of single isolated tissue cells. J Natl Cancer Inst. 1948 Dec;9(3):229–246. [PubMed] [Google Scholar]
  47. Saleh M., Barlett P. F. Evidence from neuron/lymphoma heterokaryons for a common trans-acting factor suppressing Thy-1 expression. J Neuroimmunol. 1989 Aug;23(3):203–214. doi: 10.1016/0165-5728(89)90052-0. [DOI] [PubMed] [Google Scholar]
  48. Saltiel A. R., Cuatrecasas P. Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycolipid. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5793–5797. doi: 10.1073/pnas.83.16.5793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Saltiel A. R., Fox J. A., Sherline P., Cuatrecasas P. Insulin-stimulated hydrolysis of a novel glycolipid generates modulators of cAMP phosphodiesterase. Science. 1986 Aug 29;233(4767):967–972. doi: 10.1126/science.3016898. [DOI] [PubMed] [Google Scholar]
  50. Saltiel A. R., Sherline P., Fox J. A. Insulin-stimulated diacylglycerol production results from the hydrolysis of a novel phosphatidylinositol glycan. J Biol Chem. 1987 Jan 25;262(3):1116–1121. [PubMed] [Google Scholar]
  51. Seglen P. O. Inhibitors of lysosomal function. Methods Enzymol. 1983;96:737–764. doi: 10.1016/s0076-6879(83)96063-9. [DOI] [PubMed] [Google Scholar]
  52. Soloski M. J., Hood L., Stroynowski I. Qa-region class I gene expression: identification of a second class I gene, Q9, encoding a Qa-2 polypeptide. Proc Natl Acad Sci U S A. 1988 May;85(9):3100–3104. doi: 10.1073/pnas.85.9.3100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stroynowski I., Soloski M., Low M. G., Hood L. A single gene encodes soluble and membrane-bound forms of the major histocompatibility Qa-2 antigen: anchoring of the product by a phospholipid tail. Cell. 1987 Aug 28;50(5):759–768. doi: 10.1016/0092-8674(87)90334-5. [DOI] [PubMed] [Google Scholar]
  54. Struck D. K., Lennarz W. J. Evidence for the participation of saccharide-lipids in the synthesis of the oligosaccharide chain of ovalbumin. J Biol Chem. 1977 Feb 10;252(3):1007–1013. [PubMed] [Google Scholar]
  55. Takami N., Ogata S., Oda K., Misumi Y., Ikehara Y. Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane-anchoring. J Biol Chem. 1988 Feb 25;263(6):3016–3021. [PubMed] [Google Scholar]
  56. Tokumitsu S., Fishman W. H. Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence. J Histochem Cytochem. 1983 May;31(5):647–655. doi: 10.1177/31.5.6841969. [DOI] [PubMed] [Google Scholar]
  57. Trowbridge I. S., Hyman R., Mazauskas C. The synthesis and properties of T25 blycoprotein in Thy-1-negative mutant lymphoma cells. Cell. 1978 May;14(1):21–32. doi: 10.1016/0092-8674(78)90297-0. [DOI] [PubMed] [Google Scholar]
  58. Walter E. I., Roberts W. L., Rosenberry T. L., Ratnoff W. D., Medof M. E. Structural basis for variations in the sensitivity of human decay accelerating factor to phosphatidylinositol-specific phospholipase C cleavage. J Immunol. 1990 Feb 1;144(3):1030–1036. [PubMed] [Google Scholar]
  59. Waneck G. L., Sherman D. H., Calvin S., Allen H., Flavell R. A. Tissue-specific expression of cell-surface Qa-2 antigen from a transfected Q7b gene of C57BL/10 mice. J Exp Med. 1987 May 1;165(5):1358–1370. doi: 10.1084/jem.165.5.1358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  61. Yeh E. T., Reiser H., Bamezai A., Rock K. L. TAP transcription and phosphatidylinositol linkage mutants are defective in activation through the T cell receptor. Cell. 1988 Mar 11;52(5):665–674. doi: 10.1016/0092-8674(88)90404-7. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES