Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Feb 2;128(4):525–536. doi: 10.1083/jcb.128.4.525

The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I

PMCID: PMC2199895  PMID: 7860628

Abstract

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response protein.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

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

  1. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Ballou L., Cohen R. E., Ballou C. E. Saccharomyces cerevisiae mutants that make mannoproteins with a truncated carbohydrate outer chain. J Biol Chem. 1980 Jun 25;255(12):5986–5991. [PubMed] [Google Scholar]
  4. Barnes G., Hansen W. J., Holcomb C. L., Rine J. Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step. Mol Cell Biol. 1984 Nov;4(11):2381–2388. doi: 10.1128/mcb.4.11.2381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barriocanal J. G., Bonifacino J. S., Yuan L., Sandoval I. V. Biosynthesis, glycosylation, movement through the Golgi system, and transport to lysosomes by an N-linked carbohydrate-independent mechanism of three lysosomal integral membrane proteins. J Biol Chem. 1986 Dec 15;261(35):16755–16763. [PubMed] [Google Scholar]
  6. 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]
  7. Bulleid N. J., Bassel-Duby R. S., Freedman R. B., Sambrook J. F., Gething M. J. Cell-free synthesis of enzymically active tissue-type plasminogen activator. Protein folding determines the extent of N-linked glycosylation. Biochem J. 1992 Aug 15;286(Pt 1):275–280. doi: 10.1042/bj2860275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crimaudo C., Hortsch M., Gausepohl H., Meyer D. I. Human ribophorins I and II: the primary structure and membrane topology of two highly conserved rough endoplasmic reticulum-specific glycoproteins. EMBO J. 1987 Jan;6(1):75–82. doi: 10.1002/j.1460-2075.1987.tb04721.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Enenkel C., Lehmann H., Kipper J., Gückel R., Hilt W., Wolf D. H. PRE3, highly homologous to the human major histocompatibility complex-linked LMP2 (RING12) gene, codes for a yeast proteasome subunit necessary for the peptidylglutamyl-peptide hydrolyzing activity. FEBS Lett. 1994 Mar 21;341(2-3):193–196. doi: 10.1016/0014-5793(94)80455-9. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Gibson R., Schlesinger S., Kornfeld S. The nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures. J Biol Chem. 1979 May 10;254(9):3600–3607. [PubMed] [Google Scholar]
  12. Guan J. L., Machamer C. E., Rose J. K. Glycosylation allows cell-surface transport of an anchored secretory protein. Cell. 1985 Sep;42(2):489–496. doi: 10.1016/0092-8674(85)90106-0. [DOI] [PubMed] [Google Scholar]
  13. Harnik-Ort V., Prakash K., Marcantonio E., Colman D. R., Rosenfeld M. G., Adesnik M., Sabatini D. D., Kreibich G. Isolation and characterization of cDNA clones for rat ribophorin I: complete coding sequence and in vitro synthesis and insertion of the encoded product into endoplasmic reticulum membranes. J Cell Biol. 1987 Apr;104(4):855–863. doi: 10.1083/jcb.104.4.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Helenius A. How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. Mol Biol Cell. 1994 Mar;5(3):253–265. doi: 10.1091/mbc.5.3.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jackson B. J., Kukuruzinska M. A., Robbins P. Biosynthesis of asparagine-linked oligosaccharides in Saccharomyces cerevisiae: the alg2 mutation. Glycobiology. 1993 Aug;3(4):357–364. doi: 10.1093/glycob/3.4.357. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. Kreibich G., Ulrich B. L., Sabatini D. D. Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes. J Cell Biol. 1978 May;77(2):464–487. doi: 10.1083/jcb.77.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Kuo C. L., Campbell J. L. Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation. Mol Cell Biol. 1983 Oct;3(10):1730–1737. doi: 10.1128/mcb.3.10.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Marcantonio E. E., Grebenau R. C., Sabatini D. D., Kreibich G. Identification of ribophorins in rough microsomal membranes from different organs of several species. Eur J Biochem. 1982 May;124(1):217–222. doi: 10.1111/j.1432-1033.1982.tb05928.x. [DOI] [PubMed] [Google Scholar]
  30. McGinnes L. W., Morrison T. G. The role of the individual cysteine residues in the formation of the mature, antigenic HN protein of Newcastle disease virus. Virology. 1994 May 1;200(2):470–483. doi: 10.1006/viro.1994.1210. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Riederer M. A., Hinnen A. Removal of N-glycosylation sites of the yeast acid phosphatase severely affects protein folding. J Bacteriol. 1991 Jun;173(11):3539–3546. doi: 10.1128/jb.173.11.3539-3546.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Roberts C. J., Pohlig G., Rothman J. H., Stevens T. H. Structure, biosynthesis, and localization of dipeptidyl aminopeptidase B, an integral membrane glycoprotein of the yeast vacuole. J Cell Biol. 1989 Apr;108(4):1363–1373. doi: 10.1083/jcb.108.4.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  37. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schloss P., Hermans-Borgmeyer I., Betz H., Gundelfinger E. D. Neuronal acetylcholine receptors in Drosophila: the ARD protein is a component of a high-affinity alpha-bungarotoxin binding complex. EMBO J. 1988 Sep;7(9):2889–2894. doi: 10.1002/j.1460-2075.1988.tb03146.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sikorski R. S., Boeke J. D. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 1991;194:302–318. doi: 10.1016/0076-6879(91)94023-6. [DOI] [PubMed] [Google Scholar]
  40. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Stevens T., Esmon B., Schekman R. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell. 1982 Sep;30(2):439–448. doi: 10.1016/0092-8674(82)90241-0. [DOI] [PubMed] [Google Scholar]
  43. Tsao Y. S., Ivessa N. E., Adesnik M., Sabatini D. D., Kreibich G. Carboxy terminally truncated forms of ribophorin I are degraded in pre-Golgi compartments by a calcium-dependent process. J Cell Biol. 1992 Jan;116(1):57–67. doi: 10.1083/jcb.116.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 1993 Apr;3(2):97–130. doi: 10.1093/glycob/3.2.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Winther J. R., Stevens T. H., Kielland-Brandt M. C. Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity. Eur J Biochem. 1991 May 8;197(3):681–689. doi: 10.1111/j.1432-1033.1991.tb15959.x. [DOI] [PubMed] [Google Scholar]
  46. te Heesen S., Aebi M. The genetic interaction of kar2 and wbp1 mutations. Distinct functions of binding protein BiP and N-linked glycosylation in the processing pathway of secreted proteins in Saccharomyces cerevisiae. Eur J Biochem. 1994 Jun 1;222(2):631–637. doi: 10.1111/j.1432-1033.1994.tb18906.x. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. te Heesen S., Rauhut R., Aebersold R., Abelson J., Aebi M., Clark M. W. An essential 45 kDa yeast transmembrane protein reacts with anti-nuclear pore antibodies: purification of the protein, immunolocalization and cloning of the gene. Eur J Cell Biol. 1991 Oct;56(1):8–18. [PubMed] [Google Scholar]
  50. 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]

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

RESOURCES