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. 1995 Oct 16;14(20):4949–4960. doi: 10.1002/j.1460-2075.1995.tb00178.x

STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo.

R Zufferey 1, R Knauer 1, P Burda 1, I Stagljar 1, S te Heesen 1, L Lehle 1, M Aebi 1
PMCID: PMC394598  PMID: 7588624

Abstract

N-linked glycosylation is a ubiquitous protein modification, and is essential for viability in eukaryotic cells. A lipid-linked core-oligosaccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase (OTase) complex. Based on the synthetic lethal phenotype of double mutations affecting the assembly of the lipid-linked core-oligosaccharide and the OTase activity, we have performed a novel screen for mutants in Saccharomyces cerevisiae with altered N-linked glycosylation. Besides novel mutants deficient in the assembly of the lipid-linked oligosaccharide (alg mutants), we identified the STT3 locus as being required for OTase activity in vivo. The essential STT3 protein is approximately 60% identical in amino acid sequence to its human homologue. A mutation in the STT3 locus affects substrate specificity of the OTase complex in vivo and in vitro. In stt3-3 cells very little glycosyl transfer occurs from incomplete lipid-linked oligosaccharide, whereas the transfer of full-length Glc3Man9GlcNAc2 is hardly affected as compared with wild-type cells. Depletion of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex.

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Selected References

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

  1. Abeijon C., Hirschberg C. B. Topography of glycosylation reactions in the endoplasmic reticulum. Trends Biochem Sci. 1992 Jan;17(1):32–36. doi: 10.1016/0968-0004(92)90424-8. [DOI] [PubMed] [Google Scholar]
  2. Bender A., Pringle J. R. Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Mar;11(3):1295–1305. doi: 10.1128/mcb.11.3.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  4. Breuer W., Bause E. Oligosaccharyl transferase is a constitutive component of an oligomeric protein complex from pig liver endoplasmic reticulum. Eur J Biochem. 1995 Mar 15;228(3):689–696. [PubMed] [Google Scholar]
  5. Cacan R., Labiau O., Mir A. M., Verbert A. Effect of cell attachment and growth on the synthesis and fate of dolichol-linked oligosaccharides in Chinese hamster ovary cells. Eur J Biochem. 1993 Aug 1;215(3):873–881. doi: 10.1111/j.1432-1033.1993.tb18105.x. [DOI] [PubMed] [Google Scholar]
  6. Costigan C., Gehrung S., Snyder M. A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth. Mol Cell Biol. 1992 Mar;12(3):1162–1178. doi: 10.1128/mcb.12.3.1162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gavel Y., von Heijne G. Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. Protein Eng. 1990 Apr;3(5):433–442. doi: 10.1093/protein/3.5.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Harford J. B., Waechter C. J. Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein. Arch Biochem Biophys. 1979 Oct 15;197(2):424–435. doi: 10.1016/0003-9861(79)90264-9. [DOI] [PubMed] [Google Scholar]
  9. Hasilik A., Tanner W. Carbohydrate moiety of carboxypeptidase Y and perturbation of its biosynthesis. Eur J Biochem. 1978 Nov 15;91(2):567–575. doi: 10.1111/j.1432-1033.1978.tb12710.x. [DOI] [PubMed] [Google Scholar]
  10. Heesen S., Lehle L., Weissmann A., Aebi M. Isolation of the ALG5 locus encoding the UDP-glucose:dolichyl-phosphate glucosyltransferase from Saccharomyces cerevisiae. Eur J Biochem. 1994 Aug 15;224(1):71–79. doi: 10.1111/j.1432-1033.1994.tb19996.x. [DOI] [PubMed] [Google Scholar]
  11. Herscovics A., Orlean P. Glycoprotein biosynthesis in yeast. FASEB J. 1993 Apr 1;7(6):540–550. doi: 10.1096/fasebj.7.6.8472892. [DOI] [PubMed] [Google Scholar]
  12. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kelleher D. J., Gilmore R. The Saccharomyces cerevisiae oligosaccharyltransferase is a protein complex composed of Wbp1p, Swp1p, and four additional polypeptides. J Biol Chem. 1994 Apr 29;269(17):12908–12917. [PubMed] [Google Scholar]
  17. Kelleher D. J., Kreibich G., Gilmore R. Oligosaccharyltransferase activity is associated with a protein complex composed of ribophorins I and II and a 48 kd protein. Cell. 1992 Apr 3;69(1):55–65. doi: 10.1016/0092-8674(92)90118-v. [DOI] [PubMed] [Google Scholar]
  18. Klis F. M. Review: cell wall assembly in yeast. Yeast. 1994 Jul;10(7):851–869. doi: 10.1002/yea.320100702. [DOI] [PubMed] [Google Scholar]
  19. Knauer R., Lehle L. The N-oligosaccharyltransferase complex from yeast. FEBS Lett. 1994 May 9;344(1):83–86. doi: 10.1016/0014-5793(94)00356-4. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Kranz J. E., Holm C. Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6629–6633. doi: 10.1073/pnas.87.17.6629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kukuruzinska M. A., Bergh M. L., Jackson B. J. Protein glycosylation in yeast. Annu Rev Biochem. 1987;56:915–944. doi: 10.1146/annurev.bi.56.070187.004411. [DOI] [PubMed] [Google Scholar]
  23. Kumar V., Heinemann F. S., Ozols J. Purification and characterization of avian oligosaccharyltransferase. Complete amino acid sequence of the 50-kDa subunit. J Biol Chem. 1994 May 6;269(18):13451–13457. [PubMed] [Google Scholar]
  24. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  25. Lehle L. Biosynthesis of the core region of yeast mannoproteins. Formation of a glucosylated dolichol-bound oligosaccharide precursor, its transfer to protein and subsequent modification. Eur J Biochem. 1980 Aug;109(2):589–601. doi: 10.1111/j.1432-1033.1980.tb04832.x. [DOI] [PubMed] [Google Scholar]
  26. Levin D. E., Bartlett-Heubusch E. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J Cell Biol. 1992 Mar;116(5):1221–1229. doi: 10.1083/jcb.116.5.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Levin D. E., Errede B. The proliferation of MAP kinase signaling pathways in yeast. Curr Opin Cell Biol. 1995 Apr;7(2):197–202. doi: 10.1016/0955-0674(95)80028-x. [DOI] [PubMed] [Google Scholar]
  28. Murphy L. A., Spiro R. G. Transfer of glucose to oligosaccharide-lipid intermediates by thyroid microsomal enzymes and its relationship to the N-glycosylation of proteins. J Biol Chem. 1981 Jul 25;256(14):7487–7494. [PubMed] [Google Scholar]
  29. Muñoz M. D., Hernández L. M., Basco R., Andaluz E., Larriba G. Glycosylation of yeast exoglucanase sequons in alg mutants deficient in the glucosylation steps of the lipid-linked oligosaccharide. Presence of glucotriose unit in Dol-PP-GlcNAc2Man9Glc3 influences both glycosylation efficiency and selection of N-linked sites. Biochim Biophys Acta. 1994 Dec 15;1201(3):361–366. doi: 10.1016/0304-4165(94)90063-9. [DOI] [PubMed] [Google Scholar]
  30. Paravicini G., Cooper M., Friedli L., Smith D. J., Carpentier J. L., Klig L. S., Payton M. A. The osmotic integrity of the yeast cell requires a functional PKC1 gene product. Mol Cell Biol. 1992 Nov;12(11):4896–4905. doi: 10.1128/mcb.12.11.4896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pathak R., Hendrickson T. L., Imperiali B. Sulfhydryl modification of the yeast Wbp1p inhibits oligosaccharyl transferase activity. Biochemistry. 1995 Apr 4;34(13):4179–4185. doi: 10.1021/bi00013a005. [DOI] [PubMed] [Google Scholar]
  32. Pathak R., Parker C. S., Imperiali B. The essential yeast NLT1 gene encodes the 64 kDa glycoprotein subunit of the oligosaccharyl transferase. FEBS Lett. 1995 Apr 3;362(2):229–234. doi: 10.1016/0014-5793(95)00253-6. [DOI] [PubMed] [Google Scholar]
  33. Roemer T., Paravicini G., Payton M. A., Bussey H. Characterization of the yeast (1-->6)-beta-glucan biosynthetic components, Kre6p and Skn1p, and genetic interactions between the PKC1 pathway and extracellular matrix assembly. J Cell Biol. 1994 Oct;127(2):567–579. doi: 10.1083/jcb.127.2.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rothblatt J., Schekman R. A hitchhiker's guide to analysis of the secretory pathway in yeast. Methods Cell Biol. 1989;32:3–36. doi: 10.1016/s0091-679x(08)61165-6. [DOI] [PubMed] [Google Scholar]
  35. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  36. Runge K. W., Huffaker T. C., Robbins P. W. Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway. J Biol Chem. 1984 Jan 10;259(1):412–417. [PubMed] [Google Scholar]
  37. Runge K. W., Robbins P. W. A new yeast mutation in the glucosylation steps of the asparagine-linked glycosylation pathway. Formation of a novel asparagine-linked oligosaccharide containing two glucose residues. J Biol Chem. 1986 Nov 25;261(33):15582–15590. [PubMed] [Google Scholar]
  38. Sanglard D., Sengstag C., Seghezzi W. Probing the membrane topology of Candida tropicalis cytochrome P450. Eur J Biochem. 1993 Sep 1;216(2):477–485. doi: 10.1111/j.1432-1033.1993.tb18166.x. [DOI] [PubMed] [Google Scholar]
  39. Sengstag C., Stirling C., Schekman R., Rine J. Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mol Cell Biol. 1990 Feb;10(2):672–680. doi: 10.1128/mcb.10.2.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sharma C. B., Lehle L., Tanner W. N-Glycosylation of yeast proteins. Characterization of the solubilized oligosaccharyl transferase. Eur J Biochem. 1981 May;116(1):101–108. doi: 10.1111/j.1432-1033.1981.tb05306.x. [DOI] [PubMed] [Google Scholar]
  41. Silberstein S., Collins P. G., Kelleher D. J., Rapiejko P. J., Gilmore R. The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I. J Cell Biol. 1995 Feb;128(4):525–536. doi: 10.1083/jcb.128.4.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Silberstein S., Kelleher D. J., Gilmore R. The 48-kDa subunit of the mammalian oligosaccharyltransferase complex is homologous to the essential yeast protein WBP1. J Biol Chem. 1992 Nov 25;267(33):23658–23663. [PubMed] [Google Scholar]
  43. Stagljar I., te Heesen S., Aebi M. New phenotype of mutations deficient in glucosylation of the lipid-linked oligosaccharide: cloning of the ALG8 locus. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5977–5981. doi: 10.1073/pnas.91.13.5977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Staneloni R. J., Ugalde R. A., Leloir L. F. Addition of glucose to dolichyl diphosphate oligosaccharide and transfer to protein. Eur J Biochem. 1980 Apr;105(2):275–278. doi: 10.1111/j.1432-1033.1980.tb04498.x. [DOI] [PubMed] [Google Scholar]
  45. Struhl K., Davis R. W. Transcription of the his3 gene region in Saccharomyces cerevisiae. J Mol Biol. 1981 Nov 5;152(3):535–552. doi: 10.1016/0022-2836(81)90267-9. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Trimble R. B., Byrd J. C., Maley F. Effect of glucosylation of lipid intermediates on oligosaccharide transfer in solubilized microsomes from Saccharomyces cerevisiae. J Biol Chem. 1980 Dec 25;255(24):11892–11895. [PubMed] [Google Scholar]
  48. Turco S. J., Stetson B., Robbins P. W. Comparative rates of transfer of lipid-linked oligosaccharides to endogenous glycoprotein acceptors in vitro. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4411–4414. doi: 10.1073/pnas.74.10.4411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Verostek M. F., Atkinson P. H., Trimble R. B. Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. I. Role of glucose in the initial glycosylation of invertase in the endoplasmic reticulum. J Biol Chem. 1993 Jun 5;268(16):12095–12103. [PubMed] [Google Scholar]
  50. Verostek M. F., Atkinson P. H., Trimble R. B. Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. II. Structure of novel Man6-10GlcNAc2 processing intermediates on secreted invertase. J Biol Chem. 1993 Jun 5;268(16):12104–12115. [PubMed] [Google Scholar]
  51. Vijayraghavan U., Company M., Abelson J. Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev. 1989 Aug;3(8):1206–1216. doi: 10.1101/gad.3.8.1206. [DOI] [PubMed] [Google Scholar]
  52. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]
  53. Wimmer C., Doye V., Grandi P., Nehrbass U., Hurt E. C. A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1. EMBO J. 1992 Dec;11(13):5051–5061. doi: 10.1002/j.1460-2075.1992.tb05612.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yoshida S., Ikeda E., Uno I., Mitsuzawa H. Characterization of a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1. Mol Gen Genet. 1992 Feb;231(3):337–344. doi: 10.1007/BF00292700. [DOI] [PubMed] [Google Scholar]
  55. te Heesen S., Janetzky B., Lehle L., Aebi M. The yeast WBP1 is essential for oligosaccharyl transferase activity in vivo and in vitro. EMBO J. 1992 Jun;11(6):2071–2075. doi: 10.1002/j.1460-2075.1992.tb05265.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. te Heesen S., Knauer R., Lehle L., Aebi M. Yeast Wbp1p and Swp1p form a protein complex essential for oligosaccharyl transferase activity. EMBO J. 1993 Jan;12(1):279–284. doi: 10.1002/j.1460-2075.1993.tb05654.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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