Figure 2. Biochemical Analysis of NPC2 Transfer-defective Mutants.

(A) [³H]Cholesterol transfer from NPC1(NTD) to liposomes as a function of NPC2. Each reaction, in a final volume of 200 μl buffer A (pH 5.5), contained ~50 pmol of WT NPC1(NTD)-LVPRGS-His8-FLAG complexed to [³H]cholesterol (222×10³ dpm/pmol), 20 μg PC liposomes, and the indicated concentration of WT or mutant NPC2-His10. After 10 min at 4°C, [³H]cholesterol transferred to liposomes was measured as described in Experimental Procedures (assay C). Each value represents percentage of [³H]cholesterol transferred. A blank value in the absence of NPC2 (7% transfer) was subtracted. The 100% value for transfer was 1.9 pmol/tube. (B) [³H]Cholesterol binding. Each reaction, in a final volume of 80 μl buffer A (pH 5.5) with 0.004% NP-40, contained 8 pmol of purified WT or mutant NPC2-His10, 1 μg BSA, and the indicated concentration of [³H]cholesterol (222×10³ dpm/pmol). After 2 hr at 4°C, bound [³H]cholesterol was measured. Each value represents total binding after subtraction of blank value (<0.1 pmol/tube).

(C) Time course of association of [³H]cholesterol to NPC2. Each reaction, in a final volume of 80 μl of buffer A (pH 5.5) with 0.004% NP-40, contained 8 pmol of WT or mutant NPC2-His10 and 200 nM [³H]cholesterol (132×10³ dpm/pmol). After incubation for indicated time at 4°C, bound [³H]cholesterol was determined. Each value represents total binding after subtraction of a blank value (<0.03 pmol/tube).

(D) Dissociation of previously bound [³H]cholesterol from NPC2 at different temperatures. Dissociation of [³H]cholesterol from [³H]cholesterol:NPC2-His10 (WT or mutant) was measured as described in Experimental Procedures. Each value represents the percentage of [³H]cholesterol remaining bound to WT or mutant NPC2 relative to zero-time value. The “100% initial binding” values at zero time for NPC2 was 0.48 (WT) and 0.26 (mutant) pmol/tube.

(E) [³H]Cholesterol transfer from NPC2 to NPC1(NTD). Each reaction, in a final volume of 200 μl buffer A (pH 5.5), contained ~40 pmol of WT or mutant NPC2-His10 complexed to [³H]cholesterol (222×10³ dpm/pmol) and the indicated concentration of WT NPC1(NTD)-LVPRGS-His8-FLAG. After 10 min at 4°C, [³H]cholesterol transferred was measured as described in Experimental Procedures (assay B). Each value represents percentage of [³H]cholesterol transferred to NPC1(NTD). Blank values in the absence of NPC1(NTD) (0.1–0.5% transfer) were subtracted. The 100% values for transfer from WT, V81A, and V81D NPC2 were 0.24, 0.78, and 0.29 pmol/tube, respectively (A–E) Each value is the average of duplicate assays.