Fig. 4.

Mutant NPC2 fails to transfer [³H]cholesterol from NPC1(NTD) to acceptor PC liposomes. (A) Saturation curves for equilibrium binding of [³H]cholesterol for wild-type and mutant NPC2. Binding reactions were carried out as described in Fig. 1F except that each reaction was incubated for 2 h at 4°C and contained 8 pmol of wild-type (●) or P120S mutant (○) version of NPC2-His-10. Each value is the average of duplicate assays and represents binding after subtraction of blank values (0.01–0.11 pmol). (Inset) Coomassie Brilliant blue R-250 stain of wild-type and mutant NPC2 proteins after electrophoresis on 13% SDS/PAGE (3 μg of each protein loaded on gel). (B) Transfer of [³H]cholesterol from donor NPC1(NTD) to acceptor liposomes as a function of varying concentrations of wild-type or mutant NPC2. Assays were carried out as in Fig. 3 except that each reaction was carried out for 10 min at 4°C and contained ≈50 pmol of NPC1(NTD)-LVPRGS-His-8-FLAG complexed to [³H]cholesterol (1.2 pmol; 132 × 10³ dpm/pmol), 60 μg of PC liposomes, and the indicated concentration of wild-type (●) or P120S mutant (○) version of NPC2-His-10. Each value is the average of duplicate assays and represents the percentage of [³H]cholesterol transferred to liposomes. The 100% value for transfer from NPC1(NTD) was 1.2 pmol. Blank values in the absence of NPC2 protein (8%) were subtracted.