Figure 6.
FABP7 transfers cholesterol analog in vitro. (A) Schematic illustration of in vitro dehydroergosterol (DHE) transport assay. Donor liposomes (Ld) are made of 92.5 mol% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 5 mol% DHE and 2.5 mol% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl (DNS-PE). Acceptor liposomes (La) only contain DOPC. Ld and La were incubated at 25°C for 3 min and FRET between DHE and DNS-PE (λEx: 310 nm, λEm: 525 nm) was recorded. The purified proteins were then added to a final concentration of 0.5 µM and incubated for 12 min. The transport of DHE from Ld to La is determined by the diminution in FRET between DHE and DNS-PE. (B and C) The effect of increasing concentrations of cholesterol (0, 5, 10 or 20 mol%) in donor liposomes on DHE transport by FABP7. Arrow indicates when FABP7 protein was added. Initial transport rates were quantified in C. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test. Compared with the donor liposomes without cholesterol. (D and E) Comparison of the effects of purified FABP7 and ORP2-ORD proteins on DHE transfer. Arrow indicates when the indicated proteins were added. Initial transport rates were quantified in E. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test. Compared with initial transport rates of DHE by FABP7 protein. (F and G) Comparison of the effects of FABP7 WT and mutant proteins on DHE transfer. Arrow indicates when the indicated proteins were added. Initial transport rates were quantified in G. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test. Compared with initial transport rates of DHE by wild-type (WT) FABP7 protein. (H and I) Liposome aggregation in F was assessed by dynamic light scattering. The acceptor liposomes (La, containing DOPC only, 50 µM total lipids) were mixed with the donor liposomes (Ld, containing 92.5 mol% DOPC, 5 mol% DHE, and 2.5 mol% DNS-PE, 50 µM total lipids). The size distribution of the initial liposome suspension was assessed by acquiring a first set of about 12 autocorrelation curves. The indicated proteins were then added manually and mixed thoroughly. (H) The mean radius over time. Arrow indicates when the indicated proteins were added. (I) The size distribution before (gray) and after the reaction (colored).

FABP7 transfers cholesterol analog in vitro. (A) Schematic illustration of in vitro dehydroergosterol (DHE) transport assay. Donor liposomes (Ld) are made of 92.5 mol% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 5 mol% DHE and 2.5 mol% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl (DNS-PE). Acceptor liposomes (La) only contain DOPC. Ld and La were incubated at 25°C for 3 min and FRET between DHE and DNS-PE (λEx: 310 nm, λEm: 525 nm) was recorded. The purified proteins were then added to a final concentration of 0.5 µM and incubated for 12 min. The transport of DHE from Ld to La is determined by the diminution in FRET between DHE and DNS-PE. (B and C) The effect of increasing concentrations of cholesterol (0, 5, 10 or 20 mol%) in donor liposomes on DHE transport by FABP7. Arrow indicates when FABP7 protein was added. Initial transport rates were quantified in C. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test. Compared with the donor liposomes without cholesterol. (D and E) Comparison of the effects of purified FABP7 and ORP2-ORD proteins on DHE transfer. Arrow indicates when the indicated proteins were added. Initial transport rates were quantified in E. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test. Compared with initial transport rates of DHE by FABP7 protein. (F and G) Comparison of the effects of FABP7 WT and mutant proteins on DHE transfer. Arrow indicates when the indicated proteins were added. Initial transport rates were quantified in G. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test. Compared with initial transport rates of DHE by wild-type (WT) FABP7 protein. (H and I) Liposome aggregation in F was assessed by dynamic light scattering. The acceptor liposomes (La, containing DOPC only, 50 µM total lipids) were mixed with the donor liposomes (Ld, containing 92.5 mol% DOPC, 5 mol% DHE, and 2.5 mol% DNS-PE, 50 µM total lipids). The size distribution of the initial liposome suspension was assessed by acquiring a first set of about 12 autocorrelation curves. The indicated proteins were then added manually and mixed thoroughly. (H) The mean radius over time. Arrow indicates when the indicated proteins were added. (I) The size distribution before (gray) and after the reaction (colored).

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