Liposome preparation was performed as described26 (link),27 (link),62 (link). Basic liposomes consist of 79.7% PC (L-α-phosphatidylcholine), 20% PS (L-α-phosphatidylserine) and, 0.3% Rhodamine-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt) (molar ratios). Liposomes containing an endosome lipid composition contain 31% PC, 11% SM (N-stearoyl-D-erythro-sphingosylphosphorylcholine), 14% PE (L-α-phosphatidylethanolamine), 3% PS, 40% cholesterol, 1% PtdIns (L-α-phosphatidylinositol) and 0.3% Rhodamine-PE. When PtdIns(3)P was included, the PC concentration was reduced accordingly.
The protein-to-phospholipid molar ratio was adjusted to that of early endosomes24 (link),63 (link). Accordingly, for EE-SNARE PL, the protein-to-lipid ratio of syntaxin 13, vti1a, syntaxin 6, Vamp4 was 1:2,000, 1:10,000, 1:1,200, 1:15,400, respectively. For Rab5, we quantified Rab5 and syntaxin 13 in enriched early endosomes prepared from HeLa cells (see above) by quantitative Western blot analysis using purified proteins as a standard, yielding a molar ratio of syntaxin 13: Rab5 (1:0.11), resulting in 1:16,700 as the protein-to-lipid ratio of Rab5. For proteoliposomes containing only two SNAREs, a protein:phospholipid ratio of 1:2,000 was used for each SNARE protein, and for proteoliposomes containing only one SNARE, the ratio was 1:1,000. Finally, 4-LE-SNARE-PL, a ratio of 1:2,000 was used for each of the four SNARE proteins.
The protein-to-phospholipid molar ratio was adjusted to that of early endosomes24 (link),63 (link). Accordingly, for EE-SNARE PL, the protein-to-lipid ratio of syntaxin 13, vti1a, syntaxin 6, Vamp4 was 1:2,000, 1:10,000, 1:1,200, 1:15,400, respectively. For Rab5, we quantified Rab5 and syntaxin 13 in enriched early endosomes prepared from HeLa cells (see above) by quantitative Western blot analysis using purified proteins as a standard, yielding a molar ratio of syntaxin 13: Rab5 (1:0.11), resulting in 1:16,700 as the protein-to-lipid ratio of Rab5. For proteoliposomes containing only two SNAREs, a protein:phospholipid ratio of 1:2,000 was used for each SNARE protein, and for proteoliposomes containing only one SNARE, the ratio was 1:1,000. Finally, 4-LE-SNARE-PL, a ratio of 1:2,000 was used for each of the four SNARE proteins.
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