Fig. 2.

Transfer of [³H]cholesterol from donor NPC protein to acceptor NPC protein. Schematic diagrams of the two transfer assays are shown at the top of each column. (A–D) Time course at different temperatures. Each reaction, in a final volume of 100 μl of buffer B (pH 5.5) with 0.004% Nonidet P-40, contained ≈40 pmol of NPC1(NTD)-LVPR (A and C) or NPC2-FLAG (B and D) each complexed to [³H]cholesterol (1.3–1.9 pmol; 132 × 10³ dpm/pmol) and 200 pmol of NPC1(NTD)-LVPRGS-His-8-FLAG (○) or NPC2-His-10 (●). After incubation for indicated time at 4°C (A and B) or 37°C (C and D), the amount of [³H]cholesterol transferred to the indicated acceptor His-tagged NPC protein was measured with the Ni-NTA-agarose cholesterol transfer assay. Each value represents the percentage of [³H]cholesterol transferred. The 100% values for transfer from donor were 1.9 pmol (A), 1.5 (B and C), and 1.3 (D). (E and F) Transfer at 4°C as a function of the amount of acceptor NPC protein. This experiment was carried out as in A–D except that the amount of acceptor NPC protein varied as indicated and time of incubation at 4°C was 30 min. The 100% values for transfer of [³H]cholesterol from NPC1(NTD) and NPC2 were 2.0 and 1.2 pmol, respectively. (G and H) Transfer at 4°C as a function of pH. Each reaction in a final volume of 300 μl of buffer A, B, or E with 0.004% Nonidet P-40 at the indicated pH, contained ≈30 pmol of NPC1(NTD)-LVPR (G) or NPC2-FLAG (H) each complexed to [³H]cholesterol (1.6 and 1.0 pmol, respectively) and 200 pmol of either NPC1(NTD)-LVPRGS-His-8-FLAG (○) or NPC2-His-10 (●). After incubation for 30 min at 4°C, the amount of [³H]cholesterol transferred to the indicated acceptor His-tagged NPC was measured as described above except samples were diluted with 1.1 ml of buffer A with 0.004% Nonidet P-40. Each value is the average of duplicate assays and represents the percentage of [³H]cholesterol transferred. The 100% values for transfer from NPC1(NTD) and NPC2 were 1.6 and 1.0 pmol, respectively.