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. 1994 Mar 29;91(7):2723–2727. doi: 10.1073/pnas.91.7.2723

Cloning and analysis of the Saccharomyces cerevisiae MNN9 and MNN1 genes required for complex glycosylation of secreted proteins.

C L Yip 1, S K Welch 1, F Klebl 1, T Gilbert 1, P Seidel 1, F J Grant 1, P J O'Hara 1, V L MacKay 1
PMCID: PMC43442  PMID: 8146181

Abstract

Proteins secreted by the yeast Saccharomyces cerevisiae are usually modified by the addition at asparagine-linked glycosylation sites of large heterogeneous mannan units that are highly immunogenic. Secreted proteins from mnn1 mnn9 mutant strains, in contrast, have homogeneous Man10GlcNAc2 oligosaccharides that lack the immunogenic alpha 1,3-mannose linkages. We have cloned and sequenced the MNN9 and MNN1 genes, both of which encode proteins with the characteristics of type II membrane proteins. Mnn9p is a membrane-associated protein with unknown function that is required for the addition of the long alpha 1,6-mannose backbone of the complex mannan, whereas Mnn1p is most likely the alpha 1,3-mannosyltransferase located in the Golgi apparatus.

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Selected References

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

  1. Alvarado E., Ballou L., Hernandez L. M., Ballou C. E. Localization of alpha 1----3-linked mannoses in the N-linked oligosaccharides of Saccharomyces cerevisiae mnn mutants. Biochemistry. 1990 Mar 13;29(10):2471–2482. doi: 10.1021/bi00462a006. [DOI] [PubMed] [Google Scholar]
  2. Ballou C. E. Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol. 1990;185:440–470. doi: 10.1016/0076-6879(90)85038-p. [DOI] [PubMed] [Google Scholar]
  3. Ballou L., Alvarado E., Tsai P. K., Dell A., Ballou C. E. Protein glycosylation defects in the Saccharomyces cerevisiae mnn7 mutant class. Support for the stop signal proposed for regulation of outer chain elongation. J Biol Chem. 1989 Jul 15;264(20):11857–11864. doi: 10.1016/S0021-9258(18)80145-4. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Ballou L., Hitzeman R. A., Lewis M. S., Ballou C. E. Vanadate-resistant yeast mutants are defective in protein glycosylation. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3209–3212. doi: 10.1073/pnas.88.8.3209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boer P., Steyn-Parvé E. P. Isolation and purification of an acid phosphatase from baker's yeast (Saccharomyces cerevisiae). Biochim Biophys Acta. 1966 Nov 15;128(2):400–402. doi: 10.1016/0926-6593(66)90189-5. [DOI] [PubMed] [Google Scholar]
  7. Broach J. R. Construction of high copy yeast vectors using 2-microns circle sequences. Methods Enzymol. 1983;101:307–325. doi: 10.1016/0076-6879(83)01024-1. [DOI] [PubMed] [Google Scholar]
  8. Cohen R. E., Zhang W., Ballou C. E. Effects of mannoprotein mutations on Saccharomyces cerevisiae core oligosaccharide structure. J Biol Chem. 1982 May 25;257(10):5730–5737. [PubMed] [Google Scholar]
  9. Eisenberg D., Schwarz E., Komaromy M., Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. doi: 10.1016/0022-2836(84)90309-7. [DOI] [PubMed] [Google Scholar]
  10. Gascón S., Lampen J. O. Purification of the internal invertase of yeast. J Biol Chem. 1968 Apr 10;243(7):1567–1572. [PubMed] [Google Scholar]
  11. Gopal P. K., Ballou C. E. Regulation of the protein glycosylation pathway in yeast: structural control of N-linked oligosaccharide elongation. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8824–8828. doi: 10.1073/pnas.84.24.8824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Graham T. R., Emr S. D. Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant. J Cell Biol. 1991 Jul;114(2):207–218. doi: 10.1083/jcb.114.2.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hernández L. M., Ballou L., Alvarado E., Gillece-Castro B. L., Burlingame A. L., Ballou C. E. A new Saccharomyces cerevisiae mnn mutant N-linked oligosaccharide structure. J Biol Chem. 1989 Jul 15;264(20):11849–11856. [PubMed] [Google Scholar]
  14. 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]
  15. Häusler A., Ballou L., Ballou C. E., Robbins P. W. Yeast glycoprotein biosynthesis: MNT1 encodes an alpha-1,2-mannosyltransferase involved in O-glycosylation. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6846–6850. doi: 10.1073/pnas.89.15.6846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Häusler A., Robbins P. W. Glycosylation in Saccharomyces cerevisiae: cloning and characterization of an alpha-1,2-mannosyltransferase structural gene. Glycobiology. 1992 Feb;2(1):77–84. doi: 10.1093/glycob/2.1.77. [DOI] [PubMed] [Google Scholar]
  17. Jones E. W. Proteinase mutants of Saccharomyces cerevisiae. Genetics. 1977 Jan;85(1):23–33. doi: 10.1093/genetics/85.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kanik-Ennulat C., Neff N. Vanadate-resistant mutants of Saccharomyces cerevisiae show alterations in protein phosphorylation and growth control. Mol Cell Biol. 1990 Mar;10(3):898–909. doi: 10.1128/mcb.10.3.898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Klebl F., Tanner W. Molecular cloning of a cell wall exo-beta-1,3-glucanase from Saccharomyces cerevisiae. J Bacteriol. 1989 Nov;171(11):6259–6264. doi: 10.1128/jb.171.11.6259-6264.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klein P., Kanehisa M., DeLisi C. The detection and classification of membrane-spanning proteins. Biochim Biophys Acta. 1985 May 28;815(3):468–476. doi: 10.1016/0005-2736(85)90375-x. [DOI] [PubMed] [Google Scholar]
  21. Koerner T. J., Hill J. E., Myers A. M., Tzagoloff A. High-expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors. Methods Enzymol. 1991;194:477–490. doi: 10.1016/0076-6879(91)94036-c. [DOI] [PubMed] [Google Scholar]
  22. Lewis M. S., Ballou C. E. Separation and characterization of two alpha 1,2-mannosyltransferase activities from Saccharomyces cerevisiae. J Biol Chem. 1991 May 5;266(13):8255–8261. [PubMed] [Google Scholar]
  23. MacKay V. L., Welch S. K., Insley M. Y., Manney T. R., Holly J., Saari G. C., Parker M. L. The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin. Proc Natl Acad Sci U S A. 1988 Jan;85(1):55–59. doi: 10.1073/pnas.85.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mohana Rao J. K., Argos P. A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta. 1986 Jan 30;869(2):197–214. doi: 10.1016/0167-4838(86)90295-5. [DOI] [PubMed] [Google Scholar]
  25. Nagasu T., Shimma Y., Nakanishi Y., Kuromitsu J., Iwama K., Nakayama K., Suzuki K., Jigami Y. Isolation of new temperature-sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation. Yeast. 1992 Jul;8(7):535–547. doi: 10.1002/yea.320080705. [DOI] [PubMed] [Google Scholar]
  26. Nakajima T., Ballou C. E. Yeast manno-protein biosynthesis: solubilization and selective assay of four mannosyltransferases. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3912–3916. doi: 10.1073/pnas.72.10.3912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nakanishi-Shindo Y., Nakayama K., Tanaka A., Toda Y., Jigami Y. Structure of the N-linked oligosaccharides that show the complete loss of alpha-1,6-polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae. J Biol Chem. 1993 Dec 15;268(35):26338–26345. [PubMed] [Google Scholar]
  28. Nakayama K., Nagasu T., Shimma Y., Kuromitsu J., Jigami Y. OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides. EMBO J. 1992 Jul;11(7):2511–2519. doi: 10.1002/j.1460-2075.1992.tb05316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Paulson J. C., Colley K. J. Glycosyltransferases. Structure, localization, and control of cell type-specific glycosylation. J Biol Chem. 1989 Oct 25;264(30):17615–17618. [PubMed] [Google Scholar]
  30. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Raschke W. C., Kern K. A., Antalis C., Ballou C. E. Genetic control of yeast mannan structure. Isolation and characterization of mannan mutants. J Biol Chem. 1973 Jul 10;248(13):4660–4666. [PubMed] [Google Scholar]
  32. 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]
  33. Schultz L. D., Tanner J., Hofmann K. J., Emini E. A., Condra J. H., Jones R. E., Kieff E., Ellis R. W. Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus. Gene. 1987;54(1):113–123. doi: 10.1016/0378-1119(87)90353-2. [DOI] [PubMed] [Google Scholar]
  34. Shaper N. L., Hollis G. F., Douglas J. G., Kirsch I. R., Shaper J. H. Characterization of the full length cDNA for murine beta-1,4-galactosyltransferase. Novel features at the 5'-end predict two translational start sites at two in-frame AUGs. J Biol Chem. 1988 Jul 25;263(21):10420–10428. [PubMed] [Google Scholar]
  35. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  36. Smith R. A., Duncan M. J., Moir D. T. Heterologous protein secretion from yeast. Science. 1985 Sep 20;229(4719):1219–1224. doi: 10.1126/science.3939723. [DOI] [PubMed] [Google Scholar]
  37. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Trimble R. B., Atkinson P. H. Structure of yeast external invertase Man8-14GlcNAc processing intermediates by 500-megahertz 1H NMR spectroscopy. J Biol Chem. 1986 Jul 25;261(21):9815–9824. [PubMed] [Google Scholar]
  40. Weinstein J., Lee E. U., McEntee K., Lai P. H., Paulson J. C. Primary structure of beta-galactoside alpha 2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor. J Biol Chem. 1987 Dec 25;262(36):17735–17743. [PubMed] [Google Scholar]
  41. Ziegler F. D., Maley F., Trimble R. B. Characterization of the glycosylation sites in yeast external invertase. II. Location of the endo-beta-N-acetylglucosaminidase H-resistant sequons. J Biol Chem. 1988 May 25;263(15):6986–6992. [PubMed] [Google Scholar]

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