Figure 5. Restoration of cholesterol transport to ∆NTD sequences requires that the NTD of NPC1 localize to lysosomes and bind cholesterol.

On day 0, ∆NTD cells were set up and transfected on day one as described in Figure 4 with the indicated amount of one of the following plasmids: pcDNA3.1 (control) NPC1 (A), pNTD-TM1 (B), pNTD-TM13 (C), or pNTD*-TM1 (D). After incubation for 24 hr, cells were switched to medium A with 5% LPDS containing 50 µM sodium compactin and 50 µM sodium mevalonate. On day 3, the cells received fresh medium B containing compactin and mevalonate in the presence of either 5% LPDS or 10% FCS. After incubation for 4 hr at 37°C, each cell monolayer was pulse-labeled for 2 hr with 0.1 mM sodium [¹⁴C]oleate (9019 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [¹⁴C]oleate and [¹⁴C]triglycerides. Each bar indicates the mean of duplicate incubations with individual values shown. The mean cellular content of [¹⁴C]triglycerides in the presence of FCS was not significantly different in cells transfected with pNPC1, pNTD-TM1, pNTD-T13, and pNTD*-TM1 (13.3, 12.7, 12.3, and 13.3 nmol per hr/mg protein, respectively). The bottom panel shows immunoblots of whole cell extracts (40 µg/lane) using 0.36 µg/ml of rabbit monoclonal anti-NPC1 and 1.8 µg/ml of mouse monoclonal anti-NPC2. ‡ denotes the endogenous, stably transfected ∆NTD.