Skip to main content
SpringerLink
Log in

Golgi localization of glycosyltransferases requires Gpp74p in Schizosaccharomyces pombe

  • Applied microbial and cell physiology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The majority of Golgi glycosyltransferases are type II membrane proteins with a small cytosolic tail at their N-terminus. Several mechanisms for localizing these glycosyltransferases to the Golgi have been proposed. In Saccharomyces cerevisiae, the phosphatidylinositol-4-phosphate-binding protein ScVps74p interacts with the cytosolic tail of a Golgi glycosyltransferase and contributes to its localization. In this study, we investigated whether a similar mechanism functions in the fission yeast Schizosaccharomyces pombe. First, we identified gpp74+ (GPP34 domain-containing Vps74 homolog protein), a gene encoding the S. pombe homolog of S. cerevisiae Vps74p. Deletion of the gpp74+ gene resulted in the missorting of three Golgi glycosyltransferases, SpOch1p, SpMnn9p, and SpOmh1p, to vacuoles, but not SpAnp1p, indicating Gpp74p is required for targeting some glycosyltransferases to the Golgi apparatus. Gpp74p with an N-terminal GFP-tag localized to both the Golgi apparatus and the cytosol. Golgi localization of Gpp74p was dependent on the phosphatidylinositol 4-kinase SpPik1p. Site-directed mutagenesis of hydrophobic and basic amino acids in the cytosolic tails of SpOch1p and SpMnn9p resulted in their missorting to vacuoles, indicating these cytosolic N-terminal residues are important for localization in the Golgi. Unexpectedly, no prominent alternations in protein glycosylation were observed in S. pombe gpp74Δ cells, probably due to the residual Golgi localization of some SpOch1p and SpMnn9p in these cells. Collectively, these results demonstrate that both Gpp74p-dependent and Gpp74p-independent mechanisms are responsible for the Golgi localization of glycosyltransferases to the Golgi in S. pombe.

Key points

• Gpp74p is involved in the localization of glycosyltransferases to the Golgi.

• The cytosolic tails of glycosyltransferases are important for Golgi localization.

• Gpp74p localizes to the Golgi in a SpPik1p-dependent manner.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Alfa C, Fantes P, Hyams J, Mcleod M, Warbrick E (2003) Experiments with fission yeast: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Bonangelino CJ, Chavez EM, Bonifacino JS (2002) Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae. Mol Biol Cell 13:2486–2501

    CAS  PubMed  PubMed Central  Google Scholar 

  • Burda P, Aebi M (1999) The dolichol pathway of N-linked glycosylation. Biochim Biophys Acta 1426:239–257

    CAS  PubMed  Google Scholar 

  • Cai Y, Deng Y, Horenkamp F, Reinisch KM, Burd CG (2014) Sac1-Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus. J Cell Biol 206:485–491

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cosson P, Perrin J, Bonifacino JS (2013) Anchors aweigh: protein localization and transport mediated by transmembrane domains. Trends Cell Biol 23:511–517

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dicker M, Strasser R (2015) Using glyco-engineering to produce therapeutic proteins. Expert Opin Biol Ther 15:1501–1516

    PubMed  PubMed Central  Google Scholar 

  • Dippold HC, Ng MM, Farber-Katz SE, Lee SK, Kerr ML, Peterman MC, Sim R, Wiharto PA, Galvraith KA, Madhavarapu S, Fuchs GJ, Meerloo T, Farquhar MG, Zhou H, Field SJ (2009) GOLPH3 bridges phosphatidylinositol-4-phosphate and actomyosin to stretch and shape the Golgi to promote budding. Cell 139:337–351

    CAS  PubMed  PubMed Central  Google Scholar 

  • Eckert ES, Reckmann I, Hellwig A, Röhling S, El-Battari A, Wieland FT, Popoff V (2014) Golgi phosphoprotein 3 triggers signal-mediated incorporation of glycosyltransferases into coatomer-coated (COPI) vesicles. J Biol Chem 289:31319–31329

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fukunaga T, Tanaka N, Furumoto T, Nakakita S, Ohashi T, Higuchi Y, Maekawa H, Takegawa K (2020) Characterization of N- and O-linked galactosylated oligosaccharides from fission yeast species. J Biosci Bioeng 130:128–136. https://doi.org/10.1016/j.jbiosc.2020.03.008

    Article  CAS  PubMed  Google Scholar 

  • Gleeson PA (1998) Targeting of proteins to the Golgi apparatus. Histochem Cell Biol 109:517–532

    CAS  PubMed  PubMed Central  Google Scholar 

  • Graham TR, Krasnov VA (1995) Sorting of yeast α1,3 mannosyltransferase is mediated by a luminal domain interaction, and a transmembrane domain signal that can confer clathrin-dependent Golgi localization to a secreted protein. Mol Biol Cell 6:809–824

    CAS  PubMed  PubMed Central  Google Scholar 

  • Graham TR, Seeger M, Payne GS, MacKay VL, Emr SD (1994) Clathrin-dependent localization of α1,3 mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae. J Cell Biol 127:667–678

    CAS  PubMed  Google Scholar 

  • Hancock LC, Behta RP, Lopes JM (2006) Genomic analysis of the Opi− phenotype. Genetics 173:621–634

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hase S (1994) High-performance liquid chromatography of pyridylaminated saccharides. Methods Enzymol 230:225–237

    CAS  PubMed  Google Scholar 

  • Hase S, Ikenaka T, Matsushima Y (1978) Structure analysis of oligosaccharides by tagging of the reducing end sugars with a fluorescent compound. Biochem Biophys Res Commun 85:257–263

    CAS  PubMed  Google Scholar 

  • Hausjell J, Schendl D, Weissensteiner J, Molitor C, Halbwirth H, Spadiut O (2019) Recombinant production of a hard-to-express membrane-bound cytochrome P450 in different yeasts—comparison of physiology and productivity. Yeast 37:217–226

    Google Scholar 

  • Ikeda Y, Ohashi T, Tanaka N, Takegawa K (2009) Identification and characterization of a gene required for α1,2-mannose extension in the O-linked glycan synthesis pathway in Schizosaccharomyces pombe. FEMS Yeast Res 9:115–125

    CAS  PubMed  Google Scholar 

  • Iwaki T, Hosomi A, Tokudomi S, Kusunoki Y, Fujita Y, Giga-Hama Y, Tanaka N, Takegawa K (2006) Vacuolar protein sorting receptor in Schizosaccharomyces pombe. Microbiology 152:1523–1532

    CAS  PubMed  Google Scholar 

  • Karhinen L, Makarow M (2004) Activity of recycling Golgi mannosyltransferase in the yeast endoplasmic reticulum. J Cell Sci 117:351–358

    CAS  PubMed  Google Scholar 

  • Kitajima T, Chiba Y, Jigami Y (2006) Saccharomyces cerevisiae α1,6-mannosyltransferase has a catalytic potential to transfer a second mannose molecule. FEBS J 273:5074–5085

    CAS  PubMed  Google Scholar 

  • Losev E, Reinke CA, Jelen J, Strongin DE, Bevis BJ, Glick BS (2006) Golgi maturation visualized in living yeast. Nature 441:1002–1006

    CAS  PubMed  Google Scholar 

  • Lyer P, Bhave M, Jain BK, RoyChowdhury S, Bhattacharyya D (2018) Vps74p controls Golgi size in an Arf-dependent manner. FEBS Lett 592:3720–3735

    Google Scholar 

  • Matsuura-Tokita K, Takeuchi M, Ichihara A, Mikuriya K, Nakano A (2006) Live imaging of yeast Golgi cisternal maturation. Nature 441:1007–1010

    CAS  PubMed  Google Scholar 

  • Matsuyama A, Arai R, Yashiroda Y, Shirai A, Kamata A, Sekido S, Kobayashi Y, Hashimoto A, Hamamoto M, Hiraoka Y, Horinouchi S, Yoshida M (2006) ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol 24:841–847

    CAS  PubMed  Google Scholar 

  • Milland J, Taylor SG, Dodson HC, McKenzie IF, Sandrin MS (2001) The cytoplasmic tail of α1,2-fucosyltransferase contains a sequence for Golgi localization. J Biol Chem 276:12012–12018

    CAS  PubMed  Google Scholar 

  • Milland J, Russell SM, Dodson HC, McKenzie IF, Sandrin MS (2002) The cytoplasmic tail of α1,3-galactosyltransferase inhibits Golgi localization of the full-lenghth enzyme. J Biol Chem 276:12012–12018

    Google Scholar 

  • Morita T, Takegawa K (2004) A simple and efficient procedure for transformation of Schizosaccharomyces pombe. Yeast 21:613–617

    CAS  PubMed  Google Scholar 

  • Munro S (1998) Localization of proteins to the Golgi apparatus. Trends Cell Biol 8:11–15

    CAS  PubMed  PubMed Central  Google Scholar 

  • Noda Y, Yoda K (2010) Svp26 facilitates endoplasmic reticulum to Golgi transport of a set of mannosyltransferases in Saccharomyces cerevisiae. J Biol Chem 285:15420–15429

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ohashi T, Nakakita S, Sumiyoshi W, Yamada N, Ikeda Y, Tanaka N, Takegawa K (2011) Structural analysis of α1,3-linked galactose-containing oligosaccharides in Schizosaccharomyces pombe mutants harboring single and multiple α-galactosyltransferase genes disruptions. Glycobiology 21:340–351

    CAS  PubMed  Google Scholar 

  • Ohashi T, Tanaka T, Tanaka N, Takegawa K (2020) SpMnn9p and SpAnp1p form a protein complex involved in mannan synthesis in the fission yeast Schizosaccharomyces pombe. J Biosci Bioeng. https://doi.org/10.1016/j.jbiosc.2020.06.003

  • Okamoto M, Yoko-o T, Miyakawa T, Jigami Y (2008) Cytoplasmic region of α-1,6-mannnosyltransferase Mnn9p is crucial for retrograde transport from Golgi apparatus to the endoplasmic reticulum in Saccharomyces cerevisiae. Eukaryot Cell 7:310–318

    CAS  PubMed  Google Scholar 

  • Okazaki K, Okazaki N, Kume K, Jinno S, Tanaka K, Okayama H (1990) High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe. Nucleic Acids Res 18:6485–6489

    CAS  PubMed  PubMed Central  Google Scholar 

  • Opat AS, Houghton F, Gleeson PA (2001) Steady-state localization of a medial-Golgi glycosyltransferase involves transit through the trans-Golgi network. Biochem J 358:33–40

    CAS  PubMed  PubMed Central  Google Scholar 

  • Park JS, Stenbach SK, Desautels M, Hemmingsen SM (2009) Essential role for Schizosaccharomyces pombe pik1 in septation. PLoS One 4:e6179

    PubMed  PubMed Central  Google Scholar 

  • Puthenveedu MA, Linstedt AD (2005) Subcompartmentalizing the Golgi apparatus. Curr Opin Cell Biol 17:369–375

    CAS  PubMed  Google Scholar 

  • Rabouille C, Klumperman J (2005) Opinion: the maturing role of COPI vesicles in intra-Golgi transport. Nat Rev Mol Cell Biol 6:812–817

    CAS  PubMed  Google Scholar 

  • Rodionov D, Romero PA, Berghuis AM, Herscovics A (2009) Expression and purification of recombinant M-Pol1 from Saccharomyces cerevisiae with α-1,6 mannosyltransferase activity. Protein Expr Purif 66:1–6

    CAS  PubMed  Google Scholar 

  • Schmitz KR, Liu J, Li S, Setty TG, Wood CS, Burd CG, Ferguson KM (2008) Golgi localization of glycosyltransferases requires a Vps74p oligomer. Dev Cell 14:523–534

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sugihara A, Nguyen LG, Shamin HM, Iida T, Nakase M, Takegawa K, Senda M, Jida S, Ueno M (2018) Mutation in fission yeast phosphatidylinositol 4-kinase Pik1 is synthetically lethal with defect in telomere protection protein Pot1. Biochem Biophys Res Commun 496:1284–1290

    CAS  PubMed  Google Scholar 

  • Tabuchi M, Tanaka N, Iwahara S, Takegawa K (1997) The Schizosaccharomyces pombe gms1+ gene encodes an UDP-galactose transporter homologue required for protein galactosylation. Biochem Biophys Res Commun 232:121–125

    CAS  PubMed  Google Scholar 

  • 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 53:227–235

    CAS  PubMed  Google Scholar 

  • Tanaka N, Takegawa K (2001) Functional characterization of Gms1p/UDP-galactose transporter in Schizosaccharomyces pombe mutant. Yeast 18:745–757

    CAS  PubMed  Google Scholar 

  • Tanaka N, Konomi M, Osumi M, Takegawa K (2001) Characterization of Schizosaccharomyces pombe mutant deficient in UDP-galactose transporter activity. Yeast 18:903–914

    CAS  PubMed  Google Scholar 

  • Tu L, Tai WC, Chen L, Banfield DK (2008) Signal-mediated dynamic retention of glycosyltransferases in the Golgi. Science 321:404–407

    CAS  PubMed  Google Scholar 

  • Tu L, Chen L, Banfield DK (2012) A conserved N-terminal arginine-motif in GOLPH3-family proteins mediates binding to coatomer. Traffic 13:1496–1507

    CAS  PubMed  Google Scholar 

  • Varki A (2017) Biological roles of glycans. Glycobiology 27:3–49

    CAS  PubMed  Google Scholar 

  • Wood CS, Schmitz KR, Nessman NJ, Setty TG, Ferguson KM, Burd CG (2009) PtdIns4P recognition by Vps74/GOLPH3 links PtdIns 4-kinase signaling to retrograde Golgi trafficking. J Cell Biol 187:967–975

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wood CS, Hung C-S, Huoh Y-S, Mousley CJ, Stefan CJ, Bankaitis V, Ferguson KM, Burd CG (2012) Local control of phosphatidylinositol 4-phosphate signaling in the Golgi apparatus by Vps74 and Sac1 phosphoinositide phosphatase. Mol Biol Cell 23:2527–2536

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yoko-o T, Tsukahara K, Watanabe T, Hata-Sugi N, Yoshimitsu K, Nagasu T, Jigami Y (2001) Schizosaccharomyces pombe och1+ encodes α-1,6-mannosyltransferase that is involved in outer chain elongation of N-linked oligosaccharides. FEBS Lett 489:75–80

    CAS  PubMed  Google Scholar 

  • Zhu B, Cai G, Hall EO, Freeman GJ (2007) In-Fusion™ assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques 43:354–359

    CAS  PubMed  Google Scholar 

Download references

Funding

This work was supported, in part, 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 and Industry (METI) of Japan, the New Energy and Industrial Technology Development Organization (NEDO), and JSPS KAKENHI grant number JP17H03966 (K.T.).

Author information

Author notes

  1. Takao Ohashi

    Present address: Department of Life Science, Faculty of Science and Engineering, Setsunan University, 17-8 Ikeda-Nakamachi, Neyagawa, Osaka, 572-8508, Japan

Authors and Affiliations

  1. Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan

    Takao Ohashi, Seijirou Hegi, Takamasa Fukunaga & Kaoru Takegawa

  2. ASPEX Division, Research Center, Asahi Glass Co., Ltd., Yokohama, Kanagawa, 221-8755, Japan

    Takao Ohashi

  3. Department of Agriculture, Graduate School of Science and Technology, Shinshu University, Nagano, Kamiina, 399-4598, Japan

    Akira Hosomi

Authors
  1. Takao Ohashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Seijirou Hegi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takamasa Fukunaga
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Akira Hosomi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kaoru Takegawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.T. conceived and designed the research. T.O., S.H., and T.F. conducted the experiments. T.O., A.H., and K.T. analyzed the data. T.O., S.H., and K.T. wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Kaoru Takegawa.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 767 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ohashi, T., Hegi, S., Fukunaga, T. et al. Golgi localization of glycosyltransferases requires Gpp74p in Schizosaccharomyces pombe. Appl Microbiol Biotechnol 104, 8897–8909 (2020). https://doi.org/10.1007/s00253-020-10881-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-020-10881-9

Keywords

Search

Navigation