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EMBO J. 1998 Jan 15; 17(2): 423–434.
PMCID: PMC1170393
PMID: 9430634

Multi-protein complexes in the cis Golgi of Saccharomyces cerevisiae with alpha-1,6-mannosyltransferase activity.

Abstract

Anp1p, Van1p and Mnn9p constitute a family of membrane proteins required for proper Golgi function in Saccharomyces cerevisiae. We demonstrate that these proteins colocalize within the cis Golgi, and that they are physically associated in two distinct complexes, both of which contain Mnn9p. Furthermore, we identify two new proteins in the Anp1p-Mnn9p-containing complex which have homology to known glycosyltransferases. Both protein complexes have alpha-1, 6-mannosyltransferase activity, forming a series of poly-mannose structures. These reaction products also contain some alpha-1, 2-linked mannose residues. Our data suggest that these two multi-protein complexes are responsible for the synthesis and initial branching of the long alpha-1,6-linked backbone of the hypermannose structure attached to many yeast glycoproteins.

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Selected References

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  • Abeijon C, Yanagisawa K, Mandon EC, Häusler A, Moremen K, Hirschberg CB, Robbins PW. Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. J Cell Biol. 1993 Jul;122(2):307–323. [PMC free article] [PubMed] [Google Scholar]
  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed] [Google Scholar]
  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. [PMC free article] [PubMed] [Google Scholar]
  • Antebi A, Fink GR. The yeast Ca(2+)-ATPase homologue, PMR1, is required for normal Golgi function and localizes in a novel Golgi-like distribution. Mol Biol Cell. 1992 Jun;3(6):633–654. [PMC free article] [PubMed] [Google Scholar]
  • Ballou CE. Some aspects of the structure, immunochemistry, and genetic control of yeast mannans. Adv Enzymol Relat Areas Mol Biol. 1974;40(0):239–270. [PubMed] [Google Scholar]
  • Ballou CE. Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol. 1990;185:440–470. [PubMed] [Google Scholar]
  • Ballou L, Cohen RE, Ballou CE. 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]
  • Ballou L, Alvarado E, Tsai PK, Dell A, Ballou CE. 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. [PubMed] [Google Scholar]
  • Ballou L, Hernandez LM, Alvarado E, Ballou CE. Revision of the oligosaccharide structures of yeast carboxypeptidase Y. Proc Natl Acad Sci U S A. 1990 May;87(9):3368–3372. [PMC free article] [PubMed] [Google Scholar]
  • Ballou L, Hitzeman RA, Lewis MS, Ballou CE. Vanadate-resistant yeast mutants are defective in protein glycosylation. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3209–3212. [PMC free article] [PubMed] [Google Scholar]
  • Banfield DK, Lewis MJ, Pelham HR. A SNARE-like protein required for traffic through the Golgi complex. Nature. 1995 Jun 29;375(6534):806–809. [PubMed] [Google Scholar]
  • Baudin A, Ozier-Kalogeropoulos O, Denouel A, Lacroute F, Cullin C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 1993 Jul 11;21(14):3329–3330. [PMC free article] [PubMed] [Google Scholar]
  • Benton BK, Plump SD, Roos J, Lennarz WJ, Cross FR. Over-expression of S. cerevisiae G1 cyclins restores the viability of alg1 N-glycosylation mutants. Curr Genet. 1996 Jan;29(2):106–113. [PubMed] [Google Scholar]
  • Boehm J, Ulrich HD, Ossig R, Schmitt HD. Kex2-dependent invertase secretion as a tool to study the targeting of transmembrane proteins which are involved in ER-->Golgi transport in yeast. EMBO J. 1994 Aug 15;13(16):3696–3710. [PMC free article] [PubMed] [Google Scholar]
  • Carpita NC, Gibeaut DM. Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 1993 Jan;3(1):1–30. [PubMed] [Google Scholar]
  • Chapman RE, Munro S. The functioning of the yeast Golgi apparatus requires an ER protein encoded by ANP1, a member of a new family of genes affecting the secretory pathway. EMBO J. 1994 Oct 17;13(20):4896–4907. [PMC free article] [PubMed] [Google Scholar]
  • Chappell TG, Hajibagheri MA, Ayscough K, Pierce M, Warren G. Localization of an alpha 1,2 galactosyltransferase activity to the Golgi apparatus of Schizosaccharomyces pombe. Mol Biol Cell. 1994 May;5(5):519–528. [PMC free article] [PubMed] [Google Scholar]
  • 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] [Google Scholar]
  • Cunningham KW, Wickner WT. Yeast KEX2 protease and mannosyltransferase I are localized to distinct compartments of the secretory pathway. Yeast. 1989 Jan-Feb;5(1):25–33. [PubMed] [Google Scholar]
  • Dean N. Yeast glycosylation mutants are sensitive to aminoglycosides. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1287–1291. [PMC free article] [PubMed] [Google Scholar]
  • Dean N, Poster JB. Molecular and phenotypic analysis of the S. cerevisiae MNN10 gene identifies a family of related glycosyltransferases. Glycobiology. 1996 Jan;6(1):73–81. [PubMed] [Google Scholar]
  • Erickson FL, Hannig EM. Characterization of Schizosaccharomyces pombe his1 and his5 cDNAs. Yeast. 1995 Feb;11(2):157–167. [PubMed] [Google Scholar]
  • Gahmberg CG, Tolvanen M. Why mammalian cell surface proteins are glycoproteins. Trends Biochem Sci. 1996 Aug;21(8):308–311. [PubMed] [Google Scholar]
  • Gopal PK, Ballou CE. 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. [PMC free article] [PubMed] [Google Scholar]
  • Hardwick KG, Pelham HR. SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex. J Cell Biol. 1992 Nov;119(3):513–521. [PMC free article] [PubMed] [Google Scholar]
  • Hardwick KG, Lewis MJ, Semenza J, Dean N, Pelham HR. ERD1, a yeast gene required for the retention of luminal endoplasmic reticulum proteins, affects glycoprotein processing in the Golgi apparatus. EMBO J. 1990 Mar;9(3):623–630. [PMC free article] [PubMed] [Google Scholar]
  • Harris SL, Waters MG. Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport. J Cell Biol. 1996 Mar;132(6):985–998. [PMC free article] [PubMed] [Google Scholar]
  • Hashimoto H, Sakakibara A, Yamasaki M, Yoda K. Saccharomyces cerevisiae VIG9 encodes GDP-mannose pyrophosphorylase, which is essential for protein glycosylation. J Biol Chem. 1997 Jun 27;272(26):16308–16314. [PubMed] [Google Scholar]
  • Häusler A, Robbins PW. Glycosylation in Saccharomyces cerevisiae: cloning and characterization of an alpha-1,2-mannosyltransferase structural gene. Glycobiology. 1992 Feb;2(1):77–84. [PubMed] [Google Scholar]
  • Hernandez LM, Ballou L, Alvarado E, Tsai PK, Ballou CE. Structure of the phosphorylated N-linked oligosaccharides from the mnn9 and mnn10 mutants of Saccharomyces cerevisiae. J Biol Chem. 1989 Aug 15;264(23):13648–13659. [PubMed] [Google Scholar]
  • Herscovics A, Orlean P. Glycoprotein biosynthesis in yeast. FASEB J. 1993 Apr 1;7(6):540–550. [PubMed] [Google Scholar]
  • Igual JC, Johnson AL, Johnston LH. Coordinated regulation of gene expression by the cell cycle transcription factor Swi4 and the protein kinase C MAP kinase pathway for yeast cell integrity. EMBO J. 1996 Sep 16;15(18):5001–5013. [PMC free article] [PubMed] [Google Scholar]
  • Jackson P. High-resolution polyacrylamide gel electrophoresis of fluorophore-labeled reducing saccharides. Methods Enzymol. 1994;230:250–265. [PubMed] [Google Scholar]
  • Kanik-Ennulat C, Montalvo E, Neff N. Sodium orthovanadate-resistant mutants of Saccharomyces cerevisiae show defects in Golgi-mediated protein glycosylation, sporulation and detergent resistance. Genetics. 1995 Jul;140(3):933–943. [PMC free article] [PubMed] [Google Scholar]
  • Klis FM. Review: cell wall assembly in yeast. Yeast. 1994 Jul;10(7):851–869. [PubMed] [Google Scholar]
  • Kniskern PJ, Hagopian A, Burke P, Schultz LD, Montgomery DL, Hurni WM, Ip CY, Schulman CA, Maigetter RZ, Wampler DE, et al. Characterization and evaluation of a recombinant hepatitis B vaccine expressed in yeast defective for N-linked hyperglycosylation. Vaccine. 1994 Aug;12(11):1021–1025. [PubMed] [Google Scholar]
  • Kobayashi H, Shibata N, Yonezu T, Suzuki S. Structural study of phosphomannan-protein complex of Citeromyces matritensis containing beta-1,2 linkage. Application of partial acid degradation and acetolysis techniques under mild conditions. Arch Biochem Biophys. 1987 Jul;256(1):381–396. [PubMed] [Google Scholar]
  • Kobayashi H, Shibata N, Mitobe H, Ohkubo Y, Suzuki S. Structural study of phosphomannan of yeast-form cells of Candida albicans J-1012 strain with special reference to application of mild acetolysis. Arch Biochem Biophys. 1989 Aug 1;272(2):364–375. [PubMed] [Google Scholar]
  • Kornfeld R, Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. [PubMed] [Google Scholar]
  • Lapinskas PJ, Cunningham KW, Liu XF, Fink GR, Culotta VC. Mutations in PMR1 suppress oxidative damage in yeast cells lacking superoxide dismutase. Mol Cell Biol. 1995 Mar;15(3):1382–1388. [PMC free article] [PubMed] [Google Scholar]
  • Lewis MS, Ballou CE. 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]
  • Lussier M, Sdicu AM, Ketela T, Bussey H. Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment. J Cell Biol. 1995 Nov;131(4):913–927. [PMC free article] [PubMed] [Google Scholar]
  • Lussier M, Sdicu AM, Winnett E, Vo DH, Sheraton J, Düsterhöft A, Storms RK, Bussey H. Completion of the Saccharomyces cerevisiae genome sequence allows identification of KTR5, KTR6 and KTR7 and definition of the nine-membered KRE2/MNT1 mannosyltransferase gene family in this organism. Yeast. 1997 Mar 15;13(3):267–274. [PubMed] [Google Scholar]
  • McKnight GL, Cardillo TS, Sherman F. An extensive deletion causing overproduction of yeast iso-2-cytochrome c. Cell. 1981 Aug;25(2):409–419. [PubMed] [Google Scholar]
  • Mondésert G, Reed SI. BED1, a gene encoding a galactosyltransferase homologue, is required for polarized growth and efficient bud emergence in Saccharomyces cerevisiae. J Cell Biol. 1996 Jan;132(1-2):137–151. [PMC free article] [PubMed] [Google Scholar]
  • Moremen KW, Trimble RB, Herscovics A. Glycosidases of the asparagine-linked oligosaccharide processing pathway. Glycobiology. 1994 Apr;4(2):113–125. [PubMed] [Google Scholar]
  • Munro S. An investigation of the role of transmembrane domains in Golgi protein retention. EMBO J. 1995 Oct 2;14(19):4695–4704. [PMC free article] [PubMed] [Google Scholar]
  • 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. [PubMed] [Google Scholar]
  • Nakajima T, Ballou CE. Yeast manno-protein biosynthesis: solubilization and selective assay of four mannosyltransferases. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3912–3916. [PMC free article] [PubMed] [Google Scholar]
  • Nakamura S, Takasaki H, Kobayashi K, Kato A. Hyperglycosylation of hen egg white lysozyme in yeast. J Biol Chem. 1993 Jun 15;268(17):12706–12712. [PubMed] [Google Scholar]
  • 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] [Google Scholar]
  • Napier RM, Fowke LC, Hawes C, Lewis M, Pelham HR. Immunological evidence that plants use both HDEL and KDEL for targeting proteins to the endoplasmic reticulum. J Cell Sci. 1992 Jun;102(Pt 2):261–271. [PubMed] [Google Scholar]
  • Neiman AM, Mhaiskar V, Manus V, Galibert F, Dean N. Saccharomyces cerevisiae HOC1, a suppressor of pkc1, encodes a putative glycosyltransferase. Genetics. 1997 Mar;145(3):637–645. [PMC free article] [PubMed] [Google Scholar]
  • Nilsson T, Hoe MH, Slusarewicz P, Rabouille C, Watson R, Hunte F, Watzele G, Berger EG, Warren G. Kin recognition between medial Golgi enzymes in HeLa cells. EMBO J. 1994 Feb 1;13(3):562–574. [PMC free article] [PubMed] [Google Scholar]
  • Nilsson T, Rabouille C, Hui N, Watson R, Warren G. The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells. J Cell Sci. 1996 Jul;109(Pt 7):1975–1989. [PubMed] [Google Scholar]
  • Nothwehr SF, Bryant NJ, Stevens TH. The newly identified yeast GRD genes are required for retention of late-Golgi membrane proteins. Mol Cell Biol. 1996 Jun;16(6):2700–2707. [PMC free article] [PubMed] [Google Scholar]
  • Poster JB, Dean N. The yeast VRG4 gene is required for normal Golgi functions and defines a new family of related genes. J Biol Chem. 1996 Feb 16;271(7):3837–3845. [PubMed] [Google Scholar]
  • Pryer NK, Wuestehube LJ, Schekman R. Vesicle-mediated protein sorting. Annu Rev Biochem. 1992;61:471–516. [PubMed] [Google Scholar]
  • 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] [Google Scholar]
  • Schröder S, Schimmöller F, Singer-Krüger B, Riezman H. The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in alpha-COP. J Cell Biol. 1995 Nov;131(4):895–912. [PMC free article] [PubMed] [Google Scholar]
  • Silberstein S, Gilmore R. Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase. FASEB J. 1996 Jun;10(8):849–858. [PubMed] [Google Scholar]
  • Sipos G, Puoti A, Conzelmann A. Biosynthesis of the side chain of yeast glycosylphosphatidylinositol anchors is operated by novel mannosyltransferases located in the endoplasmic reticulum and the Golgi apparatus. J Biol Chem. 1995 Aug 25;270(34):19709–19715. [PubMed] [Google Scholar]
  • Trimble RB, Atkinson PH. 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]
  • Tsai PK, Frevert J, Ballou CE. Carbohydrate structure of Saccharomyces cerevisiae mnn9 mannoprotein. J Biol Chem. 1984 Mar 25;259(6):3805–3811. [PubMed] [Google Scholar]
  • Valentín E, Herrero E, Rico H, Miragall F, Sentandreu R. Cell wall mannoproteins during the population growth phases in Saccharomyces cerevisiae. Arch Microbiol. 1987 Jul;148(2):88–94. [PubMed] [Google Scholar]
  • Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 1993 Apr;3(2):97–130. [PMC free article] [PubMed] [Google Scholar]
  • Verostek MF, Trimble RB. Mannosyltransferase activities in membranes from various yeast strains. Glycobiology. 1995 Oct;5(7):671–681. [PubMed] [Google Scholar]
  • 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] [Google Scholar]
  • 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] [Google Scholar]

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