• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of geneticsGeneticsCurrent IssueInformation for AuthorsEditorial BoardSubscribeSubmit a Manuscript
Genetics. Jun 1998; 149(2): 843–856.
PMCID: PMC1460164

Involvement of protein N-glycosyl chain glucosylation and processing in the biosynthesis of cell wall beta-1,6-glucan of Saccharomyces cerevisiae.

Abstract

beta-1,6-Glucan plays a key structural role in the yeast cell wall. Of the genes involved in its biosynthesis, the activity of Cwh41p is known, i.e., the glucosidase I enzyme of protein N-chain glucose processing. We therefore examined the effects of N-chain glucosylation and processing mutants on beta-1,6-glucan biosynthesis and show that incomplete N-chain glucose processing results in a loss of beta-1,6-glucan, demonstrating a relationship between N-chain glucosylation/processing and beta-1,6-glucan biosynthesis. To explore the involvement of other N-chain-dependent events with beta-1,6-glucan synthesis, we investigated the Saccharomyces cerevisiae KRE5 and CNE1 genes, which encode homologs of the "quality control" components UDP-Glc:glycoprotein glucosyltransferase and calnexin, respectively. We show that the essential activity of Kre5p is separate from its possible role as a UDP-Glc:glycoprotein glucosyltransferase. We also observe a approximately 30% decrease in beta-1,6-glucan upon disruption of the CNE1 gene, a phenotype that is additive with other beta-1,6-glucan synthetic mutants. Analysis of the cell wall anchorage of the mannoprotein alpha-agglutinin suggests the existence of two beta-1,6-glucan biosynthetic pathways, one N-chain dependent, the other involving protein glycosylphosphatidylinositol modification.

Full Text

The Full Text of this article is available as a PDF (202K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Brown JL, Bussey H. The yeast KRE9 gene encodes an O glycoprotein involved in cell surface beta-glucan assembly. Mol Cell Biol. 1993 Oct;13(10):6346–6356. [PMC free article] [PubMed]
  • Brown JL, Kossaczka Z, Jiang B, Bussey H. A mutational analysis of killer toxin resistance in Saccharomyces cerevisiae identifies new genes involved in cell wall (1-->6)-beta-glucan synthesis. Genetics. 1993 Apr;133(4):837–849. [PMC free article] [PubMed]
  • Burda P, Aebi M. The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation. Glycobiology. 1998 May;8(5):455–462. [PubMed]
  • Burda P, te Heesen S, Brachat A, Wach A, Düsterhöft A, Aebi M. Stepwise assembly of the lipid-linked oligosaccharide in the endoplasmic reticulum of Saccharomyces cerevisiae: identification of the ALG9 gene encoding a putative mannosyl transferase. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7160–7165. [PMC free article] [PubMed]
  • Bussey H. K1 killer toxin, a pore-forming protein from yeast. Mol Microbiol. 1991 Oct;5(10):2339–2343. [PubMed]
  • Bussey H, Sacks W, Galley D, Saville D. Yeast killer plasmid mutations affecting toxin secretion and activity and toxin immunity function. Mol Cell Biol. 1982 Apr;2(4):346–354. [PMC free article] [PubMed]
  • Caro LH, Tettelin H, Vossen JH, Ram AF, van den Ende H, Klis FM. In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae. Yeast. 1997 Dec;13(15):1477–1489. [PubMed]
  • Chen DC, Yang BC, Kuo TT. One-step transformation of yeast in stationary phase. Curr Genet. 1992 Jan;21(1):83–84. [PubMed]
  • Chen MH, Shen ZM, Bobin S, Kahn PC, Lipke PN. Structure of Saccharomyces cerevisiae alpha-agglutinin. Evidence for a yeast cell wall protein with multiple immunoglobulin-like domains with atypical disulfides. J Biol Chem. 1995 Nov 3;270(44):26168–26177. [PubMed]
  • Choudhury P, Liu Y, Bick RJ, Sifers RN. Intracellular association between UDP-glucose:glycoprotein glucosyltransferase and an incompletely folded variant of alpha1-antitrypsin. J Biol Chem. 1997 May 16;272(20):13446–13451. [PubMed]
  • Cid VJ, Durán A, del Rey F, Snyder MP, Nombela C, Sánchez M. Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae. Microbiol Rev. 1995 Sep;59(3):345–386. [PMC free article] [PubMed]
  • Dijkgraaf GJ, Brown JL, Bussey H. The KNH1 gene of Saccharomyces cerevisiae is a functional homolog of KRE9. Yeast. 1996 Jun 15;12(7):683–692. [PubMed]
  • Esmon B, Esmon PC, Schekman R. Early steps in processing of yeast glycoproteins. J Biol Chem. 1984 Aug 25;259(16):10322–10327. [PubMed]
  • Esmon PC, Esmon BE, Schauer IE, Taylor A, Schekman R. Structure, assembly, and secretion of octameric invertase. J Biol Chem. 1987 Mar 25;262(9):4387–4394. [PubMed]
  • Fernández FS, Trombetta SE, Hellman U, Parodi AJ. Purification to homogeneity of UDP-glucose:glycoprotein glucosyltransferase from Schizosaccharomyces pombe and apparent absence of the enzyme fro Saccharomyces cerevisiae. J Biol Chem. 1994 Dec 2;269(48):30701–30706. [PubMed]
  • Gietz RD, Schiestl RH, Willems AR, Woods RA. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast. 1995 Apr 15;11(4):355–360. [PubMed]
  • Hebert DN, Foellmer B, Helenius A. Glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum. Cell. 1995 May 5;81(3):425–433. [PubMed]
  • Helenius A. How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. Mol Biol Cell. 1994 Mar;5(3):253–265. [PMC free article] [PubMed]
  • Hill JE, Myers AM, Koerner TJ, Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. [PubMed]
  • Hoffman CS, 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. [PubMed]
  • Huffaker TC, Robbins PW. Yeast mutants deficient in protein glycosylation. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7466–7470. [PMC free article] [PubMed]
  • Hutchins K, Bussey H. Cell wall receptor for yeast killer toxin: involvement of (1 leads to 6)-beta-D-glucan. J Bacteriol. 1983 Apr;154(1):161–169. [PMC free article] [PubMed]
  • 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]
  • Jakob CA, Burda P, te Heesen S, Aebi M, Roth J. Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo. Glycobiology. 1998 Feb;8(2):155–164. [PubMed]
  • Ou WJ, Cameron PH, Thomas DY, Bergeron JJ. Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature. 1993 Aug 26;364(6440):771–776. [PubMed]
  • Jiang B, Sheraton J, Ram AF, Dijkgraaf GJ, Klis FM, Bussey H. CWH41 encodes a novel endoplasmic reticulum membrane N-glycoprotein involved in beta 1,6-glucan assembly. J Bacteriol. 1996 Feb;178(4):1162–1171. [PMC free article] [PubMed]
  • Parlati F, Dominguez M, Bergeron JJ, Thomas DY. Saccharomyces cerevisiae CNE1 encodes an endoplasmic reticulum (ER) membrane protein with sequence similarity to calnexin and calreticulin and functions as a constituent of the ER quality control apparatus. J Biol Chem. 1995 Jan 6;270(1):244–253. [PubMed]
  • Kapteyn JC, Montijn RC, Vink E, de la Cruz J, Llobell A, Douwes JE, Shimoi H, Lipke PN, Klis FM. Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer. Glycobiology. 1996 Apr;6(3):337–345. [PubMed]
  • Petrescu AJ, Butters TD, Reinkensmeier G, Petrescu S, Platt FM, Dwek RA, Wormald MR. The solution NMR structure of glucosylated N-glycans involved in the early stages of glycoprotein biosynthesis and folding. EMBO J. 1997 Jul 16;16(14):4302–4310. [PMC free article] [PubMed]
  • Kilker RD, Jr, Saunier B, Tkacz JS, Herscovics A. Partial purification from Saccharomyces cerevisiae of a soluble glucosidase which removes the terminal glucose from the oligosaccharide Glc3Man9GlcNAc2. J Biol Chem. 1981 May 25;256(10):5299–5603. [PubMed]
  • Ram AF, Wolters A, Ten Hoopen R, Klis FM. A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast. 1994 Aug;10(8):1019–1030. [PubMed]
  • Kollár R, Reinhold BB, Petráková E, Yeh HJ, Ashwell G, Drgonová J, Kapteyn JC, Klis FM, Cabib E. Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin. J Biol Chem. 1997 Jul 11;272(28):17762–17775. [PubMed]
  • Ram AF, Brekelmans SS, Oehlen LJ, Klis FM. Identification of two cell cycle regulated genes affecting the beta 1,3-glucan content of cell walls in Saccharomyces cerevisiae. FEBS Lett. 1995 Jan 23;358(2):165–170. [PubMed]
  • Kollár R, Petráková E, Ashwell G, Robbins PW, Cabib E. Architecture of the yeast cell wall. The linkage between chitin and beta(1-->3)-glucan. J Biol Chem. 1995 Jan 20;270(3):1170–1178. [PubMed]
  • Raschke WC, Kern KA, Antalis C, Ballou CE. Genetic control of yeast mannan structure. Isolation and characterization of mannan mutants. J Biol Chem. 1973 Jul 10;248(13):4660–4666. [PubMed]
  • Kolodziej PA, Young RA. Epitope tagging and protein surveillance. Methods Enzymol. 1991;194:508–519. [PubMed]
  • Reiss G, te Heesen S, Zimmerman J, Robbins PW, Aebi M. Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway. Glycobiology. 1996 Jul;6(5):493–498. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Roemer T, Bussey H. Yeast Kre1p is a cell surface O-glycoprotein. Mol Gen Genet. 1995 Nov 15;249(2):209–216. [PubMed]
  • Lu CF, Kurjan J, Lipke PN. A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin. Mol Cell Biol. 1994 Jul;14(7):4825–4833. [PMC free article] [PubMed]
  • Roemer T, Paravicini G, Payton MA, 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. [PMC free article] [PubMed]
  • Lu CF, Montijn RC, Brown JL, Klis F, Kurjan J, Bussey H, Lipke PN. Glycosyl phosphatidylinositol-dependent cross-linking of alpha-agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall. J Cell Biol. 1995 Feb;128(3):333–340. [PMC free article] [PubMed]
  • Romero PA, Dijkgraaf GJ, Shahinian S, Herscovics A, Bussey H. The yeast CWH41 gene encodes glucosidase I. Glycobiology. 1997 Oct;7(7):997–1004. [PubMed]
  • Lussier M, Sdicu AM, Bussereau F, Jacquet M, Bussey H. The Ktr1p, Ktr3p, and Kre2p/Mnt1p mannosyltransferases participate in the elaboration of yeast O- and N-linked carbohydrate chains. J Biol Chem. 1997 Jun 13;272(24):15527–15531. [PubMed]
  • Lussier M, White AM, Sheraton J, di Paolo T, Treadwell J, Southard SB, Horenstein CI, Chen-Weiner J, Ram AF, Kapteyn JC, et al. Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics. 1997 Oct;147(2):435–450. [PMC free article] [PubMed]
  • Saunier B, Kilker RD, Jr, Tkacz JS, Quaroni A, Herscovics A. Inhibition of N-linked complex oligosaccharide formation by 1-deoxynojirimycin, an inhibitor of processing glucosidases. J Biol Chem. 1982 Dec 10;257(23):14155–14161. [PubMed]
  • Manners DJ, Masson AJ, Patterson JC. The structure of a beta-(1 leads to 3)-D-glucan from yeast cell walls. Biochem J. 1973 Sep;135(1):19–30. [PMC free article] [PubMed]
  • Schreuder MP, Brekelmans S, van den Ende H, Klis FM. Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae. Yeast. 1993 Apr;9(4):399–409. [PubMed]
  • Manners DJ, Masson AJ, Patterson JC, Björndal H, Lindberg B. The structure of a beta-(1--6)-D-glucan from yeast cell walls. Biochem J. 1973 Sep;135(1):31–36. [PMC free article] [PubMed]
  • Sharma CB, Lehle L, Tanner W. N-Glycosylation of yeast proteins. Characterization of the solubilized oligosaccharyl transferase. Eur J Biochem. 1981 May;116(1):101–108. [PubMed]
  • Meaden P, Hill K, Wagner J, Slipetz D, Sommer SS, Bussey H. The yeast KRE5 gene encodes a probable endoplasmic reticulum protein required for (1----6)-beta-D-glucan synthesis and normal cell growth. Mol Cell Biol. 1990 Jun;10(6):3013–3019. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Simons JF, Ferro-Novick S, Rose MD, Helenius A. BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast. J Cell Biol. 1995 Jul;130(1):41–49. [PMC free article] [PubMed]
  • Nuoffer C, Jenö P, Conzelmann A, Riezman H. Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane. Mol Cell Biol. 1991 Jan;11(1):27–37. [PMC free article] [PubMed]
  • Sousa MC, Ferrero-Garcia MA, Parodi AJ. Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. Biochemistry. 1992 Jan 14;31(1):97–105. [PubMed]
  • Ora A, Helenius A. Calnexin fails to associate with substrate proteins in glucosidase-deficient cell lines. J Biol Chem. 1995 Nov 3;270(44):26060–26062. [PubMed]
  • 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Ou WJ, Bergeron JJ, Li Y, Kang CY, Thomas DY. Conformational changes induced in the endoplasmic reticulum luminal domain of calnexin by Mg-ATP and Ca2+. J Biol Chem. 1995 Jul 28;270(30):18051–18059. [PubMed]
  • Wach A, Brachat A, Pöhlmann R, Philippsen P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 1994 Dec;10(13):1793–1808. [PubMed]
  • Wada I, Rindress D, Cameron PH, Ou WJ, Doherty JJ, 2nd, Louvard D, Bell AW, Dignard D, Thomas DY, Bergeron JJ. SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J Biol Chem. 1991 Oct 15;266(29):19599–19610. [PubMed]
  • Trombetta SE, Parodi AJ. Purification to apparent homogeneity and partial characterization of rat liver UDP-glucose:glycoprotein glucosyltransferase. J Biol Chem. 1992 May 5;267(13):9236–9240. [PubMed]
  • Trombetta ES, Simons JF, Helenius A. Endoplasmic reticulum glucosidase II is composed of a catalytic subunit, conserved from yeast to mammals, and a tightly bound noncatalytic HDEL-containing subunit. J Biol Chem. 1996 Nov 1;271(44):27509–27516. [PubMed]
  • Vai M, Gatti E, Lacanà E, Popolo L, Alberghina L. Isolation and deduced amino acid sequence of the gene encoding gp115, a yeast glycophospholipid-anchored protein containing a serine-rich region. J Biol Chem. 1991 Jul 5;266(19):12242–12248. [PubMed]
  • Yip CL, Welch SK, Klebl F, Gilbert T, Seidel P, Grant FJ, O'Hara PJ, MacKay VL. Cloning and analysis of the Saccharomyces cerevisiae MNN9 and MNN1 genes required for complex glycosylation of secreted proteins. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2723–2727. [PMC free article] [PubMed]
  • van Berkel MA, Caro LH, Montijn RC, Klis FM. Glucosylation of chimeric proteins in the cell wall of Saccharomyces cerevisiae. FEBS Lett. 1994 Jul 25;349(1):135–138. [PubMed]
  • Vernet T, Dignard D, Thomas DY. A family of yeast expression vectors containing the phage f1 intergenic region. Gene. 1987;52(2-3):225–233. [PubMed]

Articles from Genetics are provided here courtesy of Genetics Society of America

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...