Skip to main content
SpringerLink
Log in

Expression of Functional Myc-Tagged Conserved Oligomeric Golgi (COG) Subcomplexes in Mammalian Cells

  • Protocol
  • First Online:
Membrane Trafficking

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1270))

Abstract

Docking and fusion of transport carriers in eukaryotic cells are regulated by a family of multi-subunit tethering complexes (MTC) that sequentially and/or simultaneously interact with other components of vesicle fusion machinery, such as SNAREs, Rabs, coiled-coil tethers, and vesicle coat components. Probing for interactions of multi-protein complexes has relied heavily on the method of exogenously expressing individual proteins and then determining their interaction stringency. An obvious pitfall of this method is that the protein interactions are not occurring in their native multi-subunit state. Here, we describe an assay where we express all eight subunits of the conserved oligomeric Golgi (COG) complex that contain the same triple-Myc epitope tag and then an assay for the (sub) complex’s interaction with known protein partners. The expression of all eight proteins allows for the assembled complex to interact with partner proteins, and by having the same tag on all eight COG subunits, we are able to very accurately quantify the interaction with each subunit. The use of this assay has highlighted a very important level of specificity of interactions between COG subcomplexes and their intracellular partners.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Lupashin V, Ungar D (2008) COG complex. In: Mironov A, Pavelka M (eds) The Golgi apparatus. Springer, Wien, NY, pp 120–127

    Google Scholar 

  2. Bonifacino JS, Glick BS (2004) The mechanisms of vesicle budding and fusion. Cell 116:153–166

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. 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  CAS  PubMed  Google Scholar 

  5. 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  CAS  PubMed  Google Scholar 

  6. Foulquier F (2009) COG defects, birth and rise! Biochim Biophys Acta 1792:896–902

    Article  CAS  PubMed  Google Scholar 

  7. Glick BS, Nakano A (2009) Membrane traffic within the Golgi apparatus. Annu Rev Cell Dev Biol 25:113–132

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Cottam NP, Ungar D (2012) Retrograde vesicle transport in the Golgi. Protoplasma 249:943–955

    Article  CAS  PubMed  Google Scholar 

  9. Ungar D, Oka T, Brittle EE, Vasile E, Lupashin VV, Chatterton JE et al (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 Central  CAS  PubMed  Google Scholar 

  10. Whyte JRC, 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  CAS  PubMed  Google Scholar 

  11. 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  CAS  PubMed  Google Scholar 

  12. 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  CAS  PubMed  Google Scholar 

  13. 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  CAS  PubMed  Google Scholar 

  14. 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 Central  CAS  PubMed  Google Scholar 

  15. 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  CAS  PubMed  Google Scholar 

  16. Shestakova A, Suvorova E, Pavliv O, Khaidakova G, Lupashin V (2007) Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5a/Sed5 enhances intra-Golgi SNARE complex stability. J Cell Biol 179:1179–1192

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Sun Y, Shestakova A, Hunt L, Sehgal S, Lupashin V, Storrie B (2007) Rab6 regulates both ZW10/RINT-1 and conserved oligomeric Golgi complex-dependent Golgi trafficking and homeostasis. Mol Biol Cell 18:4129–4142

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Miller VJ, Sharma P, Kudlyk TA, Frost L, Rofe AP, Watson IJ et al (2013) Molecular insights into vesicle tethering at the Golgi by the conserved oligomeric Golgi (COG) complex and the golgin TATA element modulatory factor (TMF). J Biol Chem 288:4229–4240

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Willett R, Kudlyk T, Pokrovskaya I, Schonherr R, Ungar D, Duden R et al (2013) COG complexes form spatial landmarks for distinct SNARE complexes. Nat Commun 4:1553

    Article  PubMed Central  PubMed  Google Scholar 

  20. Sohda M, Misumi Y, Yoshimura S, Nakamura N, Fusano T, Ogata S et al (2007) The interaction of two tethering factors, p115 and COG complex, is required for Golgi integrity. Traffic 8:270–284

    Article  CAS  PubMed  Google Scholar 

  21. Sohda M, Misumi Y, Yamamoto A, Nakamura N, Ogata S, Sakisaka S et al (2010) Interaction of Golgin-84 with the COG complex mediates the intra-Golgi retrograde transport. Traffic 11:1552–1566

    Article  CAS  PubMed  Google Scholar 

  22. Willett R, Pokrovskaya I, Kudlyk T, Lupashin V (2014) Multipronged interaction of the COG complex with intracellular membranes. Cell Logist 4:e27888

    Article  PubMed Central  PubMed  Google Scholar 

  23. Laufman O, Hong W, Lev S (2011) The COG complex interacts directly with Syntaxin 6 and positively regulates endosome-to-TGN retrograde transport. J Cell Biol 194:459–472

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Kudlyk T, Willett R, Pokrovskaya ID, Lupashin V (2013) COG6 interacts with a subset of the Golgi SNAREs and is important for the Golgi complex integrity. Traffic 14:194–204

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Laufman O, Hong W, Lev S (2013) The COG complex interacts with multiple Golgi SNAREs and enhances fusogenic assembly of SNARE complexes. J Cell Sci 126:1506–1516

    Article  CAS  PubMed  Google Scholar 

  26. VanRheenen SM, Cao X, Sapperstein SK, Chiang EC, Lupashin VV, Barlowe C et al (1999) Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p. J Cell Biol 147:729–742

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. 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  CAS  PubMed  Google Scholar 

  28. Rothbauer U, Zolghadr K, Muyldermans S, Schepers A, Cardoso MC, Leonhardt H (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7:282–289

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 W. Markham, Slot 505, Little Rock, AR, 72205, USA

    Rose A. Willett, Tetyana A. Kudlyk & Vladimir V. Lupashin

Authors
  1. Rose A. Willett
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tetyana A. Kudlyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vladimir V. Lupashin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vladimir V. Lupashin .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, National University of Singapore, Singapore, Singapore

    Bor Luen Tang

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Willett, R.A., Kudlyk, T.A., Lupashin, V.V. (2015). Expression of Functional Myc-Tagged Conserved Oligomeric Golgi (COG) Subcomplexes in Mammalian Cells. In: Tang, B. (eds) Membrane Trafficking. Methods in Molecular Biology, vol 1270. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2309-0_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2309-0_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2308-3

  • Online ISBN: 978-1-4939-2309-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation