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. 1990 Nov;10(11):5796–5805. doi: 10.1128/mcb.10.11.5796

Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae.

P Orlean 1
PMCID: PMC361358  PMID: 2146492

Abstract

Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose. Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells. Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene. At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in [2-3H]myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both [2-3H]myo-inositol and [2-3H]glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis. The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein. Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C. Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature.

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Selected References

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

  1. Albright C. F., Orlean P., Robbins P. W. A 13-amino acid peptide in three yeast glycosyltransferases may be involved in dolichol recognition. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7366–7369. doi: 10.1073/pnas.86.19.7366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arnold E., Tanner W. An obligatory role of protein glycosylation in the life cycle of yeast cells. FEBS Lett. 1982 Nov 1;148(1):49–53. doi: 10.1016/0014-5793(82)81240-4. [DOI] [PubMed] [Google Scholar]
  3. Basson M. E., Thorsness M., Finer-Moore J., Stroud R. M., Rine J. Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis. Mol Cell Biol. 1988 Sep;8(9):3797–3808. doi: 10.1128/mcb.8.9.3797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beck P. J., Orlean P., Albright C., Robbins P. W., Gething M. J., Sambrook J. F. The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line. Mol Cell Biol. 1990 Sep;10(9):4612–4622. doi: 10.1128/mcb.10.9.4612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Becker G. W., Lester R. L. Biosynthesis of phosphoinositol-containing sphingolipids from phosphatidylinositol by a membrane preparation from Saccharomyces cerevisiae. J Bacteriol. 1980 Jun;142(3):747–754. doi: 10.1128/jb.142.3.747-754.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bernstein M., Kepes F., Schekman R. Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Mar;9(3):1191–1199. doi: 10.1128/mcb.9.3.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  8. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  9. Chapman A., Fujimoto K., Kornfeld S. The primary glycosylation defect in class E Thy-1-negative mutant mouse lymphoma cells is an inability to synthesize dolichol-P-mannose. J Biol Chem. 1980 May 25;255(10):4441–4446. [PubMed] [Google Scholar]
  10. Chapman A., Trowbridge I. S., Hyman R., Kornfeld S. Structure of the lipid-linked oligosaccharides that accumulate in class E Thy-1-negative mutant lymphomas. Cell. 1979 Jul;17(3):509–515. doi: 10.1016/0092-8674(79)90259-9. [DOI] [PubMed] [Google Scholar]
  11. Conzelmann A., Fankhauser C., Desponds C. Myoinositol gets incorporated into numerous membrane glycoproteins of Saccharomyces cerevisiae; incorporation is dependent on phosphomannomutase (sec53). EMBO J. 1990 Mar;9(3):653–661. doi: 10.1002/j.1460-2075.1990.tb08157.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Culbertson M. R., Henry S. A. Inositol-requiring mutants of Saccharomyces cerevisiae. Genetics. 1975 May;80(1):23–40. doi: 10.1093/genetics/80.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Doering T. L., Masterson W. J., Englund P. T., Hart G. W. Biosynthesis of the glycosyl phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein. Origin of the non-acetylated glucosamine. J Biol Chem. 1989 Jul 5;264(19):11168–11173. [PubMed] [Google Scholar]
  16. Doering T. L., Masterson W. J., Hart G. W., Englund P. T. Biosynthesis of glycosyl phosphatidylinositol membrane anchors. J Biol Chem. 1990 Jan 15;265(2):611–614. [PubMed] [Google Scholar]
  17. 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]
  18. Ferguson M. A., Homans S. W., Dwek R. A., Rademacher T. W. Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science. 1988 Feb 12;239(4841 Pt 1):753–759. doi: 10.1126/science.3340856. [DOI] [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. Ferro-Novick S., Novick P., Field C., Schekman R. Yeast secretory mutants that block the formation of active cell surface enzymes. J Cell Biol. 1984 Jan;98(1):35–43. doi: 10.1083/jcb.98.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Finne J., Krusius T., Margolis R. K., Margolis R. U. Novel mannitol-containing oligosaccharides obtained by mild alkaline borohydride treatment of a chondroitin sulfate proteoglycan from brain. J Biol Chem. 1979 Oct 25;254(20):10295–10300. [PubMed] [Google Scholar]
  22. Hirschberg C. B., Snider M. D. Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem. 1987;56:63–87. doi: 10.1146/annurev.bi.56.070187.000431. [DOI] [PubMed] [Google Scholar]
  23. Hubbard S. C., Ivatt R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu Rev Biochem. 1981;50:555–583. doi: 10.1146/annurev.bi.50.070181.003011. [DOI] [PubMed] [Google Scholar]
  24. Hubbard S. C., Robbins P. W. Synthesis of the N-linked oligosaccharides of glycoproteins. Assembly of the lipid-linked precursor oligosaccharide and its relation to protein synthesis in vivo. J Biol Chem. 1980 Dec 25;255(24):11782–11793. [PubMed] [Google Scholar]
  25. Huffaker T. C., Robbins P. W. Temperature-sensitive yeast mutants deficient in asparagine-linked glycosylation. J Biol Chem. 1982 Mar 25;257(6):3203–3210. [PubMed] [Google Scholar]
  26. Huffaker T. C., Robbins P. W. Yeast mutants deficient in protein glycosylation. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7466–7470. doi: 10.1073/pnas.80.24.7466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kepes F., Schekman R. The yeast SEC53 gene encodes phosphomannomutase. J Biol Chem. 1988 Jul 5;263(19):9155–9161. [PubMed] [Google Scholar]
  28. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Krusius T., Reinhold V. N., Margolis R. K., Margolis R. U. Structural studies on sialylated and sulphated O-glycosidic mannose-linked oligosaccharides in the chondroitin sulphate proteoglycan of brain. Biochem J. 1987 Jul 1;245(1):229–234. doi: 10.1042/bj2450229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lehrman M. A., Zeng Y. Pleiotropic resistance to glycoprotein processing inhibitors in Chinese hamster ovary cells. The role of a novel mutation in the asparagine-linked glycosylation pathway. J Biol Chem. 1989 Jan 25;264(3):1584–1593. [PubMed] [Google Scholar]
  32. Low M. G. Glycosyl-phosphatidylinositol: a versatile anchor for cell surface proteins. FASEB J. 1989 Mar;3(5):1600–1608. doi: 10.1096/fasebj.3.5.2522071. [DOI] [PubMed] [Google Scholar]
  33. Ma H., Kunes S., Schatz P. J., Botstein D. Plasmid construction by homologous recombination in yeast. Gene. 1987;58(2-3):201–216. doi: 10.1016/0378-1119(87)90376-3. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Mayor S., Menon A. K., Cross G. A. Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. II. Lipid structures of phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids. J Biol Chem. 1990 Apr 15;265(11):6174–6181. [PubMed] [Google Scholar]
  36. Menon A. K., Mayor S., Ferguson M. A., Duszenko M., Cross G. A. Candidate glycophospholipid precursor for the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoproteins. J Biol Chem. 1988 Feb 5;263(4):1970–1977. [PubMed] [Google Scholar]
  37. Normington K., Kohno K., Kozutsumi Y., Gething M. J., Sambrook J. S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell. 1989 Jun 30;57(7):1223–1236. doi: 10.1016/0092-8674(89)90059-7. [DOI] [PubMed] [Google Scholar]
  38. Orlean P., Albright C., Robbins P. W. Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein. J Biol Chem. 1988 Nov 25;263(33):17499–17507. [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Rose M. D., Fink G. R. KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell. 1987 Mar 27;48(6):1047–1060. doi: 10.1016/0092-8674(87)90712-4. [DOI] [PubMed] [Google Scholar]
  42. Rose M. D., Misra L. M., Vogel J. P. KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell. 1989 Jun 30;57(7):1211–1221. doi: 10.1016/0092-8674(89)90058-5. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Schauer I., Emr S., Gross C., Schekman R. Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme. J Cell Biol. 1985 May;100(5):1664–1675. doi: 10.1083/jcb.100.5.1664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sharma C. B., Babczinski P., Lehle L., Tanner W. The role of dolicholmonophosphate in glycoprotein biosynthesis in Saccharomyces cerevisiae. Eur J Biochem. 1974 Jul 1;46(1):35–41. doi: 10.1111/j.1432-1033.1974.tb03594.x. [DOI] [PubMed] [Google Scholar]
  46. Stoll J., Krag S. S. A mutant of Chinese hamster ovary cells with a reduction in levels of dolichyl phosphate available for glycosylation. J Biol Chem. 1988 Aug 5;263(22):10766–10773. [PubMed] [Google Scholar]
  47. Stoll J., Robbins A. R., Krag S. S. Mutant of Chinese hamster ovary cells with altered mannose 6-phosphate receptor activity is unable to synthesize mannosylphosphoryldolichol. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2296–2300. doi: 10.1073/pnas.79.7.2296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stoll J., Rosenwald A. G., Krag S. S. A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions. J Biol Chem. 1988 Aug 5;263(22):10774–10782. [PubMed] [Google Scholar]
  49. Tanner W., Lehle L. Protein glycosylation in yeast. Biochim Biophys Acta. 1987 Apr 27;906(1):81–99. doi: 10.1016/0304-4157(87)90006-2. [DOI] [PubMed] [Google Scholar]
  50. Wilson D. W., Wilcox C. A., Flynn G. C., Chen E., Kuang W. J., Henzel W. J., Block M. R., Ullrich A., Rothman J. E. A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast. Nature. 1989 Jun 1;339(6223):355–359. doi: 10.1038/339355a0. [DOI] [PubMed] [Google Scholar]
  51. 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]

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