Applied Microbiology and Biotechnology

, Volume 93, Issue 4, pp 1609–1618 | Cite as

N-glycans are not required for the efficient degradation of the mutant Saccharomyces cerevisiae CPY* in Schizosaccharomyces pombe

  • Hiroyuki Mukaiyama
  • Michiko Kodera
  • Naotaka Tanaka
  • Kaoru Takegawa
Applied genetics and molecular biotechnology


In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPY* was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPY* was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY* degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells.


Schizosaccharomyces pombe ERAD Carboxypeptidase Y Protein degradation 



This work was partly supported by the Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R & D of New Industrial Science and Technology Frontiers by the Ministry of Economy, Trade & Industry, as supported by the New Energy and Industrial Technology Development Organization.


  1. Bays NW, Gardner RG, Seelig LP, Joazeiro CA, Hampton RY (2001) Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation. Nat Cell Biol 3:24–29CrossRefGoogle Scholar
  2. Bordallo J, Plemper RK, Finger A, Wolf DH (1998) Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. Mol Biol Cell 9:209–222Google Scholar
  3. Broker M, Ragg H, Karges HE (1987) Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Biochim Bophys Acta 908:203–213Google Scholar
  4. Buschhorn BA, Kostova Z, Medicherla B, Wolf DH (2004) A genome-wide screen identifies Yos9p as essential for ER-associated degradation of glycoproteins. FEBS Lett 577:422–426CrossRefGoogle Scholar
  5. Caramelo JJ, Parodi AJ (2007) How sugars convey information on protein conformation in the endoplasmic reticulum. Semin Cell Dev Biol 18:732–742CrossRefGoogle Scholar
  6. Carvalho P, Goder V, Rapoport TA (2006) Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126:361–373CrossRefGoogle Scholar
  7. Clerc S, Hirsch C, Oggier DM, Deprez P, Jakob C, Sommer T, Aebi M (2009) Htm1 protein generates the N-glycan signal for glycoprotein degradation in the endoplasmic reticulum. J Cell Biol 184:159–172CrossRefGoogle Scholar
  8. Cox JS, Walter P (1996) A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87:391–404CrossRefGoogle Scholar
  9. D’Alessio C, Caramelo JJ, Parodi AJ (2010) UDP-GlC:glycoprotein glucosyltransferase-glucosidase II, the ying-yang of the ER quality control. Semin Cell Dev Biol 21:491–499CrossRefGoogle Scholar
  10. Denic V, Quan EM, Weissman JS (2006) A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation. Cell 126:349–359CrossRefGoogle Scholar
  11. Ellgaard L, Helenius A (2003) Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4:181–191CrossRefGoogle Scholar
  12. Fagioli C, Sitia R (2001) Glycoprotein quality control in the endoplasmic reticulum. Mannose trimming by endoplasmic reticulum mannosidase I times the proteasomal degradation of unassembled immunoglobulin subunits. J Biol Chem 276:12885–12892CrossRefGoogle Scholar
  13. Fanchiotti S, Fernández F, D’Alessio C, Parodi AJ (1998) The UDP-Glc:glycoprotein glucosyltransferase is essential for Schizosaccharomyces pombe viability under conditions of extreme endoplasmic reticulum stress. J Cell Biol 143:625–635CrossRefGoogle Scholar
  14. Fernandez F, Jannatipour M, Hellman U, Rokeach LA, Parodi AJ (1996) A new stress protein: synthesis of Schizosaccharomyces pombe UDP-GIc:glycoprotein glucosyltransferase mRNA is induced by stress conditions but the enzyme is not essential for cell viability. EMBO J 15:705–713Google Scholar
  15. Finger A, Knop M, Wolf DH (1993) Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast. Eur J Biochem 218:565–574CrossRefGoogle Scholar
  16. Gardner RG, Swarbrick GM, Bays NW, Cronin SR, Wilhovsky S, Seelig L, Kim C, Hampton RY (2000) Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrd1p by Hrd3p. J Cell Biol 151:69–82CrossRefGoogle Scholar
  17. Gauss R, Jarosch E, Sommer T, Hirsch C (2006a) A complex of Yos9p and the HRD ligase integrates endoplasmic reticulum quality control into the degradation machinery. Nat Cell Biol 8:849–854CrossRefGoogle Scholar
  18. Gauss R, Sommer T, Jarosch E (2006b) The Hrd1p ligase complex forms a linchpin between ER-lumenal substrate selection and Cdc48p recruitment. EMBO J 25:1827–1835CrossRefGoogle Scholar
  19. Giga-Hama Y, Kumagai H (1999) Expression system for foreign genes using the fission yeast Schizosaccharomyces pombe. Biotechnol Appl Biochem 30:235–244Google Scholar
  20. Giga-Hama Y, Tohda H, Takegawa K, Kumagai H (2007) Schizosaccharomyces pombe minimum genome factory. Biotechnol Appl Biocham 46:147–155CrossRefGoogle Scholar
  21. Haigh NG, Johnson AE (2002) A new role for BiP: closing the aqueous translocon pore during protein integration into the ER membrane. J Cell Biol 156:261–270CrossRefGoogle Scholar
  22. Hammond C, Braakman I, Helenius A (1994) Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control. Proc Natl Acad Sci 91:913–917CrossRefGoogle Scholar
  23. Helenius A, Aebi M (2001) Intracellular functions of N-linked glycans. Science 291:2364–2369CrossRefGoogle Scholar
  24. Hirao K, Natsuka Y, Tamura T, Wada I, Morito D, Natsuka S, Romero P, Sleno B, Tremblay LO, Herscovics A, Nagata K, Hosokawa N (2006) EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming. J Biol Chem 281:9650–9658CrossRefGoogle Scholar
  25. Hughes BT, Nwosu CC, Espenshade PJ (2009) Degradation of sterol regulatory element-binding protein precursor requires the endoplasmic reticulum-associated degradation components Ubc7 and Hrd1 in fission yeast. J Biol Chem 284:20512–20521CrossRefGoogle Scholar
  26. Huyer G, Piluek WF, Fansler Z, Kreft SG, Hochstrasser M, Brodsky JL, Michaelis S (2004) Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein. J Biol Chem 279:38369–38378CrossRefGoogle Scholar
  27. Iwaki T, Osawa F, Onishi M, Koga T, Fujita Y, Hosomi A, Tanaka N, Fukui Y, Takegawa K (2003) Characterization of vps33 +, a gene required for vacuolar biogenesis and protein sorting in Schizosaccharomyces pombe. Yeast 20:845–855CrossRefGoogle Scholar
  28. Jannatipour M, Callejo M, Parodi AJ, Armstrong J, Rokeach LA (1998) Calnexin and BiP interact with acid phosphatase independently of glucose trimming and reglucosylation in Schizosaccharomyces pombe. Biochem 37:17253–17261CrossRefGoogle Scholar
  29. Knop M, Finger A, Braun T, Hellmuth K, Wolf DH (1996a) Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J 15:753–763Google Scholar
  30. Knop M, Hauser N, Wolf DH (1996b) N-Glycosylation affects endoplasmic reticulum degradation of a mutated derivative of carboxypeptidase yscY in yeast. Yeast 12:1229–1238CrossRefGoogle Scholar
  31. Kostova Z, Wolf DH (2005) Importance of carbohydrate positioning in the recognition of mutated CPY for ER-associated degradation. J Cell Sci 118:1485–1492CrossRefGoogle Scholar
  32. Kota J, Gilstring CF, Ljungdahl PO (2007) Membrane chaperone Shr3 assists in folding amino acid permeases preventing precocious ERAD. J Cell Biol 176:617–628CrossRefGoogle Scholar
  33. Liao M, Faouzi S, Karyakin A, Correia MA (2006) Endoplasmic reticulum-associated degradation of cytochrome P450 CYP3A4 in Saccharomyces cerevisiae: further characterization of cellular participants and structural determinants. Mol Pharmacol 69:1897–1904CrossRefGoogle Scholar
  34. Lipari F, Herscovics A (1996) Role of the cysteine residues in the alpha1,2-mannosidase involved in N-glycan biosynthesis in Saccharomyces cerevisiae. The conserved Cys340 and Cys385 residues form an essential disulfide bond. J Biol Chem 271:27615–27622CrossRefGoogle Scholar
  35. Mast SW, Diekman K, Karaveg K, Davis A, Sifers RN, Moremen KW (2005) Human EDEM2, a novel homolog of family 47 glycosidases, is involved in ER-associated degradation of glycoproteins. Glycobiology 15:421–436CrossRefGoogle Scholar
  36. Morita T, Takegawa K (2004) A simple and efficient procedure for transformation of Schizosaccharomyces pombe. Yeast 21:613–617CrossRefGoogle Scholar
  37. Movsichoff F, Castro OA, Parodi AJ (2005) Characterization of Schizosaccharomyces pombe ER α-mannosidase: a reevaluation of the role of the enzyme on ER-associated degradation. Mol Biol Cell 16:4714–4724CrossRefGoogle Scholar
  38. Mukaiyama H, Tohda H, Takegawa K (2010) Overexpression of protein disulfide isomerases enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe. Appl Microbiol Biotech 86:1135–1143CrossRefGoogle Scholar
  39. Nakamura T, Nakamura-Kubo M, Hirata A, Shimoda C (2001) The Schizosaccharomyces pombe spo3+ gene is required for assembly of the forespore membrane and genetically interacts with psy1(+)-encoding syntaxin-like protein. Mol Biol Cell 12:3955–3972Google Scholar
  40. Olivari S, Galli C, Alanen H, Ruddock L, Molinari M (2005) A novel stress-induced EDEM variant regulating endoplasmic reticulum-associated glycoprotein degradation. J Biol Chem 280:2424–2428CrossRefGoogle Scholar
  41. Quan EM, Kamiya Y, Kamiya D, Denic V, Weibezahn J, Kato K, Weissman JS (2008) Defining the glycan destruction signal for endoplasmic reticulum-associated degradation. Mol Cell 32:870–877CrossRefGoogle Scholar
  42. Rapoport TA, Rolls MM, Jungnickel B (1996) Approaching the mechanism of protein transport across the ER membrane. Curr Opin Cell Biol 8:499–504CrossRefGoogle Scholar
  43. Ravid T, Kreft SG, Hochstrasser M (2006) Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways. EMBO J 25:533–543CrossRefGoogle Scholar
  44. Ruddock LW, Molinari M (2006) N-glycan processing in ER quality control. J Cell Sci 119:4373–4380CrossRefGoogle Scholar
  45. Sagt CM, Muller WH, van der Heide L, Boonstra J, Verkleij AJ, Verrips CT (2002) Impaired cutinase secretion in Saccharomyces cerevisiae induces irregular endoplasmic reticulum (ER) membrane proliferation, oxidative stress, and ER-associated degradation. Appl Environ Microbiol 68:2155–2160CrossRefGoogle Scholar
  46. Sander P, Grunewald S, Bach M, Haase W, Reilander H, Michel H (1994) Heterologous expression of the human D2S dopamine receptor in pretase-deficient Saccharomyces cerevisiae strains. Eur J Biochem 226:697–705CrossRefGoogle Scholar
  47. Simeon A, Egner R, Gascon S, Suarez-Rendueles P (1995) Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated, sorted and matured in the fission yeast Schizosaccharomyces pombe. Yeast 11:271–282CrossRefGoogle Scholar
  48. Szathmary R, Bielmann R, Nita-Lazar M, Burda P, Jakob CA (2005) Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD. Mol Cell 19:765–775CrossRefGoogle Scholar
  49. Tabuchi M, Iwaihara O, Ohtani Y, Ohuchi N, Sakurai J, Morita T, Iwahara S, Takegawa K (1997) Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe. J Bacteriol 179:4179–4189Google Scholar
  50. Takegawa K, Tohda H, Sasaki M, Idiris A, Ohashi T, Mukaiyama H, Giga-Hama Y, Kumagai H (2009) Production of heterologous proteins using the fission-yeast (Schizosaccharomyces pombe) expression system. Biotechnol Appl Biochem 53:227–235CrossRefGoogle Scholar
  51. Taxis C, Hitt R, Park SH, Deak PM, Kostova Z, Wolf DH (2003) Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD. J Biol Chem 278:35903–35913CrossRefGoogle Scholar
  52. Tommasino M, Contorni M, Scarlato V, Bugnoli M, Maundrell K, Cavalieri F (1990) Synthesis, phosphorylation, and nuclear localization of human papillomavirus E7 protein in Schizosaccharomyces pombe. Gene 93:265–270CrossRefGoogle Scholar
  53. Tsai B, Ye Y, Rapoport TA (2002) Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nat Rev Mol Cell Biol 3:246–255CrossRefGoogle Scholar
  54. Vashist S, Ng DT (2004) Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control. J Cell Biol 165:41–52CrossRefGoogle Scholar
  55. Ware FE, Vassilakos A, Peterson PA, Jackson MR, Lehrman MA, Williams DB (1995) The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. J Biol Chem 270:4697–4704CrossRefGoogle Scholar
  56. Xu X, Kanbara K, Azakami H, Kato A (2004) Expression and characterization of Saccharomyces cerevisiae Cne1p, a calnexin homologue. J Biochem 135:615–618CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Hiroyuki Mukaiyama
    • 1
    • 2
    • 3
  • Michiko Kodera
    • 2
  • Naotaka Tanaka
    • 2
  • Kaoru Takegawa
    • 2
    • 3
  1. 1.ASPEX Division, Research CenterAsahi Glass Co., LtdKanagawaJapan
  2. 2.Department of Life Sciences, Faculty of AgricultureKagawa UniversityKagawaJapan
  3. 3.Department of Bioscience and Biotechnology, Faculty of AgricultureKyushu UniversityFukuokaJapan

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