SNAREs pp 211-220 | Cite as

Determination of Sec18-Lipid Interactions by Liposome-Binding Assay

  • Matthew L. Starr
  • Rutilio FrattiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)


Protein-lipid binding interactions play a key role in the regulation of peripheral membrane protein function. Liposome-binding assays are a simple and affordable means of screening for specific protein-lipid interactions. Liposomes are prepared by mixing phospholipid combinations of interest before drying and rehydration. Sonication of the lipid mixture produces small unilamellar vesicles (SUVs) which are incubated with a protein of interest to allow for any binding to occur. Liposomes and liposome-protein complexes are floated on a sucrose gradient by centrifugation to separate them from unbound protein. Bound protein levels are easily determined by SDS-PAGE and Western blotting. This approach provides a reliable means of assaying novel protein-lipid interactions in vitro. Here we use liposome floatation to show the binding of the SNARE-activating protein Sec18 (mammalian NSF) to phosphatidic acid.

Key words

Liposome Phospholipids Membrane trafficking Membrane fusion Sec18 NSF Phosphatidic acid SNARE 



This work was supported in part by NIH grant GM101132 to RAF.


  1. 1.
    Jahn R, Lang T, Südhof TC (2003) Membrane fusion. Cell 112:519–533CrossRefGoogle Scholar
  2. 2.
    Boeddinghaus C, Merz AJ, Laage R, Ungermann C (2002) A cycle of Vam7p release from and PtdIns 3-P-dependent rebinding to the yeast vacuole is required for homotypic vacuole fusion. J Cell Biol 157:79–89CrossRefGoogle Scholar
  3. 3.
    Cabrera M, Nordmann M, Perz A, Schmedt D, Gerondopoulos A, Barr F et al (2014) The Mon1-Ccz1 GEF activates the Rab7 GTPase Ypt7 via a longin fold-Rab interface and association with PI-3-P-positive membranes. J Cell Sci 27(Pt 5):1043–1051CrossRefGoogle Scholar
  4. 4.
    Cheever ML, Sato TK, de Beer T, Kutateladze TG, Emr SD, Overduin M (2001) Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes. Nat Cell Biol 3:613–618CrossRefGoogle Scholar
  5. 5.
    Fratti RA, Jun Y, Merz AJ, Margolis N, Wickner W (2004) Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles. J Cell Biol 167:1087–1098CrossRefGoogle Scholar
  6. 6.
    Jun Y, Fratti RA, Wickner W (2004) Diacylglycerol and its formation by Phospholipase C regulate Rab- and SNARE- dependent yeast vacuole fusion. J Biol Chem 279:53186–53195CrossRefGoogle Scholar
  7. 7.
    Karunakaran S, Sasser T, Rajalekshmi S, Fratti RA (2012) SNAREs, HOPS, and regulatory lipids control the dynamics of vacuolar actin during homotypic fusion. J Cell Sci 14:1683–1692CrossRefGoogle Scholar
  8. 8.
    Karunakaran S, Fratti R (2013) The lipid composition and physical properties of the yeast vacuole affect the Hemifusion-fusion transition. Traffic 14:650–662CrossRefGoogle Scholar
  9. 9.
    Kato M, Wickner W (2001) Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion. EMBO J 20:4035–4040CrossRefGoogle Scholar
  10. 10.
    Lawrence G, Brown CC, Flood BA, Karunakaran S, Cabrera M, Nordmann M et al (2014) Dynamic association of the PI3P-interacting Mon1-Ccz1 GEF with vacuoles is controlled through its phosphorylation by the type-1 casein kinase Yck3. Mol Biol Cell 25:1608–1619CrossRefGoogle Scholar
  11. 11.
    Mayer A, Scheglmann D, Dove S, Glatz A, Wickner W, Haas A (2000) Phosphatidylinositol 4,5-bisphosphate regulates two steps of homotypic vacuole fusion. Mol Biol Cell 11:807–817CrossRefGoogle Scholar
  12. 12.
    Miner GE, Starr ML, Hurst LR, Sparks RP, Padolina M, Fratti RA (2016) The central polybasic region of the soluble SNARE (soluble N-Ethylmaleimide-sensitive factor attachment protein receptor) Vam7 affects binding to phosphatidylinositol 3-phosphate by the PX (Phox homology) domain. J Biol Chem 291:17651–17663CrossRefGoogle Scholar
  13. 13.
    Miner GE, Starr ML, Hurst LR, Fratti RA (2017) Deleting the DAG kinase Dgk1 augments yeast vacuole fusion through increased Ypt7 activity and altered membrane fluidity. Traffic 18:315–329CrossRefGoogle Scholar
  14. 14.
    Sasser T, Qiu QS, Karunakaran S, Padolina M, Reyes A, Flood B et al (2012) Yeast lipin 1 orthologue pah1p regulates vacuole homeostasis and membrane fusion. J Biol Chem 287:2221–2236CrossRefGoogle Scholar
  15. 15.
    Starr ML, Hurst LR, Fratti RA (2016) Phosphatidic acid sequesters Sec18p from cis-SNARE complexes to inhibit priming. Traffic 17:1091–1109CrossRefGoogle Scholar
  16. 16.
    Stroupe C, Collins KM, Fratti RA, Wickner W (2006) Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p. EMBO J 25:1579–1589CrossRefGoogle Scholar
  17. 17.
    Mima J, Wickner W (2009) Complex lipid requirements for SNARE-and SNARE chaperone dependent membrane fusion. J Biol Chem 284:27114–27122CrossRefGoogle Scholar
  18. 18.
    Del Vecchio K, Stahelin RV (2016) Using surface plasmon resonance to quantitatively assess lipid-protein interactions. Methods Mol Biol 1376:141–153CrossRefGoogle Scholar
  19. 19.
    Manifava M, Thuring JW, Lim ZY, Packman L, Holmes AB, Ktistakis NT (2001) Differential binding of traffic-related proteins to phosphatidic acid- or phosphatidylinositol (4,5)- bisphosphate-coupled affinity reagents. J Biol Chem 276:8987–8994CrossRefGoogle Scholar
  20. 20.
    Matsuoka K, Morimitsu Y, Uchida K, Schekman R (1998) Coat assembly directs v-SNARE concentration into synthetic COPII vesicles. Mol Cell 2:703–708CrossRefGoogle Scholar
  21. 21.
    van den Bogaart G, Meyenberg K, Diederichsen U, Jahn R (2012) Phosphatidylinositol 4,5-bisphosphate increases Ca2+ affinity of synaptotagmin-1 by 40-fold. J Biol Chem 287:16447–16453CrossRefGoogle Scholar
  22. 22.
    Kooijman EE, Tieleman DP, Testerink C, Munnik T, Rijkers DT, Burger KN et al (2007) An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins. J Biol Chem 282:11356–11364CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA

Personalised recommendations