All lipids were obtained from Avanti Polar Lipids, Inc. For t-SNARE reconstitution, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), and cholesterol were mixed in a molar ratio of 60:20:10:10. For v-SNARE reconstitution, POPC, POPE, POPS, cholesterol, N-(7-nitro-2,1,3-benzoxadiazole-4-yl)-1,2-dipalmitoyl phosphatidylethanolamine (NBD-DPPE), and N-(Lissamine rhodamine B sulfonyl)-DPPE (rhodamine-DPPE) were mixed at a molar ratio of 60:17:10:10:1.5:1.5. SNARE proteoliposomes were prepared by detergent dilution and isolated on an Accudenz density gradient flotation (Weber et al., 1998 (link)). SNARE proteins were kept at physiologically relevant densities, with protein/lipid ratios at 1:200 for v-SNAREs (similar to VAMP2 densities reported for native synaptic vesicles; Jahn and Südhof, 1994 (link); Walch-Solimena et al., 1995 (link)) and at 1:500 for t-SNARE liposomes. Reconstituted liposomes were routinely monitored by dynamic light scattering and electron microscopy with negative staining.
Protocol detail
Reconstitution of SNARE Proteoliposomes
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1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine
1 palmitoyl 2 oleoyl sn glycero 3 phosphoethanolamine
1 palmitoyl 2 oleoyl sn glycero 3 phosphoserine
Cholesterol
Detergent
Dilution
Dipalmitoyl phosphatidylethanolamine
Dppe
Electron microscopy
Lipid
Liposomes
Lissamine rhodamine b
Molar
Nbd dppe
Phosphocholine
Phosphoethanolamine
Phosphoserine
Protein
Proteoliposomes
Rhodamine
Snare
Synaptic vesicles
T snare
V snares
Vamp2
Corresponding Organization : University of Colorado Boulder
Other organizations : Yale University
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Variable analysis
independent variables
- Lipid composition (molar ratio) for t-SNARE reconstitution: POPC, POPE, POPS, and cholesterol
- Lipid composition (molar ratio) for v-SNARE reconstitution: POPC, POPE, POPS, cholesterol, NBD-DPPE, and rhodamine-DPPE
- Reconstituted SNARE proteoliposomes (their properties and characteristics)
- SNARE protein/lipid ratios: 1:200 for v-SNAREs and 1:500 for t-SNARE liposomes
- Physiologically relevant densities of SNARE proteins
- Monitoring of reconstituted liposomes by dynamic light scattering and electron microscopy with negative staining
- Not explicitly mentioned
- Not explicitly mentioned
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