Determination of Sec18-Lipid Interactions by Liposome-Binding Assay
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 wordsLiposome Phospholipids Membrane trafficking Membrane fusion Sec18 NSF Phosphatidic acid SNARE
This work was supported in part by NIH grant GM101132 to RAF.
- 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