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Fluorescent Microscopy as a Tool to Elucidate Dysfunction and Mislocalization of Golgi Glycosyltransferases in COG Complex Depleted Mammalian Cells

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Glycosyltransferases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1022))

Abstract

Staining of molecules such as proteins and glycoconjugates allows for an analysis of their localization within the cell and provides insight into their functional status. Glycosyltransferases, a class of enzymes which are responsible for glycosylating host proteins, are mostly localized to the Golgi apparatus, and their localization is maintained in part by a protein vesicular tethering complex, the conserved oligomeric Golgi (COG) complex. Here we detail a combination of fluorescent lectin and immuno-staining in cells depleted of COG complex subunits to examine the status of Golgi glycosyltransferases. The combination of these techniques allows for a detailed characterization of the changes in function and localization of Golgi glycosyltransferases with respect to transient COG subunit depletion.

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References

  1. Shorter J, Warren G (2002) Golgi architecture and inheritance. Annu Rev Cell Dev Biol 18:379–420

    Article  PubMed  CAS  Google Scholar 

  2. Pokrovskaya ID, Willett R, Smith RD, Morelle W, Kudlyk T, Lupashin VV (2011) COG complex specifically regulates the maintenance of Golgi glycosylation machinery. Glycobiology 21(12):1554–1569

    Article  PubMed  CAS  Google Scholar 

  3. Suvorova ES, Kurten RC, Lupashin VV (2001) Identification of a human orthologue of Sec34p as a component of the cis-Golgi vesicle tethering machinery. J Biol Chem 276: 22810–22818

    Article  PubMed  CAS  Google Scholar 

  4. Suvorova ES, Duden R, Lupashin VV (2002) The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins. J Cell Biol 157:631–643

    Article  PubMed  CAS  Google Scholar 

  5. Whyte JR, Munro S (2001) The Sec34/35 Golgi transport complex is related to the ­exocyst, defining a family of complexes involved in multiple steps of membrane traffic. Dev Cell 1:527–537

    Article  PubMed  CAS  Google Scholar 

  6. Ungar D, Oka T, Brittle EE, Vasile E, Lupashin VV, Chatterton JE, Heuser JE, Krieger M, Waters MG (2002) Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function. J Cell Biol 157:405–415

    Article  PubMed  CAS  Google Scholar 

  7. Ungar D, Oka T, Vasile E, Krieger M, Hughson FM (2005) Subunit architecture of the conserved oligomeric golgi complex. J Biol Chem 280:32729–32735

    Article  PubMed  CAS  Google Scholar 

  8. Fotso P, Koryakina Y, Pavliv O, Tsiomenko AB, Lupashin VV (2005) Cog1p plays a central role in the organization of the yeast conserved oligomeric golgi complex. J Biol Chem 280:27613–27623

    Article  PubMed  CAS  Google Scholar 

  9. Bruinsma P, Spelbrink RG, Nothwehr SF (2004) Retrograde transport of the mannosyltransferase Och1p to the early Golgi requires a component of the COG transport complex. J Biol Chem 279:39814–39823

    Article  PubMed  CAS  Google Scholar 

  10. Shestakova A, Zolov S, Lupashin V (2006) COG complex-mediated recycling of Golgi glycosyltransferases is essential for normal protein glycosylation. Traffic 7:191–204

    Article  PubMed  CAS  Google Scholar 

  11. Kingsley DM, Kozarsky KF, Segal M, Krieger M (1986) Three types of low density lipoprotein receptor-deficient mutant have pleiotropic defects in the synthesis of N-linked, O-linked, and lipid- linked carbohydrate chains. J Cell Biol 102:1576–1585

    Article  PubMed  CAS  Google Scholar 

  12. Reynders E, Foulquier F, Annaert W, Matthijs G (2011) How Golgi glycosylation meets and needs trafficking: the case of the COG complex. Glycobiology 21:853–863

    Article  PubMed  CAS  Google Scholar 

  13. Wu X, Steet RA, Bohorov O, Bakker J, Newell J, Krieger M, Spaapen L, Kornfeld S, Freeze HH (2004) Mutation of the COG complex subunit gene COG7 causes a lethal congenital disorder. Nat Med 10:518–523

    Article  PubMed  CAS  Google Scholar 

  14. Foulquier F, Ungar D, Reynders E, Zeevaert R, Mills P, Garcia-Silva MT, Briones P, Winchester B, Morelle W, Krieger M, Annaert W, Matthijs G (2007) A new inborn error of glycosylation due to a Cog8 deficiency reveals a critical role for the Cog1-Cog8 interaction in COG complex formation. Hum Mol Genet 16:717–730

    Article  PubMed  CAS  Google Scholar 

  15. Foulquier F, Vasile E, Schollen E, Callewaert N, Raemaekers T, Quelhas D, Jaeken J, Mills P, Winchester B, Krieger M, Annaert W, Matthijs G (2006) Conserved oligomeric Golgi complex subunit 1 deficiency reveals a previously uncharacterized congenital disorder of glycosylation type II. Proc Natl Acad Sci USA 103:3764–3769

    Article  PubMed  CAS  Google Scholar 

  16. Kranz C, Ng BG, Sun L, Sharma V, Eklund EA, Miura Y, Ungar D, Lupashin V, Winkel RD, Cipollo JF, Costello CE, Loh E, Hong W, Freeze HH (2007) COG8 deficiency causes new congenital disorder of glycosylation type IIh. Hum Mol Genet 16:731–741

    Article  PubMed  CAS  Google Scholar 

  17. Ng BG, Kranz C, Hagebeuk EE, Duran M, Abeling NG, Wuyts B, Ungar D, Lupashin V, Hartdorff CM, Poll-The BT, Freeze HH (2007) Molecular and clinical characterization of a moroccan Cog7 deficient patient. Mol Genet Metab 91:201–204

    Article  PubMed  CAS  Google Scholar 

  18. Zeevaert R, Foulquier F, Jaeken J, Matthijs G (2008) Deficiencies in subunits of the conserved oligomeric Golgi (COG) complex define a novel group of congenital disorders of glycosylation. Mol Genet Metab 93:15–21

    Article  PubMed  CAS  Google Scholar 

  19. Reynders E, Foulquier F, Leao Teles E, Quelhas D, Morelle W, Rabouille C, Annaert W, Matthijs G (2009) Golgi function and dysfunction in the first COG4-deficient CDG type II patient. Hum Mol Genet 18:3244–3256

    Article  PubMed  CAS  Google Scholar 

  20. Paesold Burda P, Maag C, Troxler H, Foulquier F, Kleinert P, Schnabel S, Baumgartner M, Hennet T (2009) Deficiency in COG5 causes a moderate form of congenital disorders of glycosylation. Hum Mol Genet 18(22): 4350–4356

    Article  PubMed  CAS  Google Scholar 

  21. Lubbehusen J, Thiel C, Rind N, Ungar D, Prinsen BH, de Koning TJ, van Hasselt PM, Korner C (2010) Fatal outcome due to deficiency of subunit 6 of the conserved oligomeric Golgi complex leading to a new type of congenital disorders of glycosylation. Hum Mol Genet 19:3623–3633

    Article  PubMed  Google Scholar 

  22. Nilsson T, Hoe MH, Slusarewicz P, Rabouille C, Watson R, Hunte F, Watzele G, Berger EG, Warren G (1994) Kin recognition between medial Golgi enzymes in HeLa cells. EMBO J 13:562–574

    PubMed  CAS  Google Scholar 

  23. Miles S, McManus H, Forsten KE, Storrie B (2001) Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block. J Cell Biol 155:543–555

    Article  PubMed  CAS  Google Scholar 

  24. Krieger M, Brown M, Goldstein J (1981) Isolation of Chinese hamster cell mutants defective in the receptor-mediated endocytosis of low density lipoprotein. J Mol Biol 150:167–184

    Article  PubMed  CAS  Google Scholar 

  25. Podos SD, Reddy P, Ashkenas J, Krieger M (1994) LDLC encodes a brefeldin A-sensitive, peripheral Golgi protein required for normal Golgi function. J Cell Biol 127:679–691

    Article  PubMed  CAS  Google Scholar 

  26. Chatterton JE, Hirsch D, Schwartz JJ, Bickel PE, Rosenberg RD, Lodish HF, Krieger M (1999) Expression cloning of LDLB, a gene essential for normal Golgi function and assembly of the ldlCp complex. Proc Natl Acad Sci USA 96:915–920

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    Rose A. Willett, Irina D. Pokrovskaya & Vladimir V. Lupashin

Authors
  1. Rose A. Willett
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  2. Irina D. Pokrovskaya
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  3. Vladimir V. Lupashin
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Editors and Affiliations

  1. Department of Medicine, Queen's University, Kingston, Ontario, Canada

    Inka Brockhausen

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Willett, R.A., Pokrovskaya, I.D., Lupashin, V.V. (2013). Fluorescent Microscopy as a Tool to Elucidate Dysfunction and Mislocalization of Golgi Glycosyltransferases in COG Complex Depleted Mammalian Cells. In: Brockhausen, I. (eds) Glycosyltransferases. Methods in Molecular Biology, vol 1022. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-465-4_6

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  • DOI: https://doi.org/10.1007/978-1-62703-465-4_6

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-464-7

  • Online ISBN: 978-1-62703-465-4

  • eBook Packages: Springer Protocols

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