(A) Domain structure of NPC1 protein. The N-terminal domain (residues 23–259 including the polyproline linker), middle lumenal domain (MLD, 372–620), and C-terminal domain (CTD, 854–1098) are colored red, blue, and yellow, respectively. (B) NPC1 residues mutated to Cys for disulfide bond formation between the polyproline linker and CTD (see inset). The location of the sterol-sensing domain is shown in orange; P691 faces the back. (C) Confocal immunofluorescence microscopy of mouse NPC1 P251C/L929C and LAMP1 proteins expressed in HeLa cells (bar, 20 μm). (D) Confocal immunofluorescence microscopy of cholesterol accumulation rescue. NPC1−/− HeLa cells were transfected with GFP-mouse NPC1-wild type or P251C/L929C plasmids for 48 h. Thirty-two hours post transfection, cells were incubated with 1 µM U18666a for 16 h; cells were briefly incubated with 10 mM methylamine hydrochloride and chased for cholesterol export for 1 h in 5% LPDS medium, followed by immediate fixation. Intrinsic GFP fluorescence and AF647-PFO* labeling are shown (bar, 20 μm). Images represent maximum intensity projections. (E) Flow cytometry of the experiment shown in (D). GFP-positive cells of similar intensity were analyzed: NPC1-/-, 497 cells; NPC1 wild type, 478 cells; P251/L929C, 1486 cells. Cell numbers were normalized for comparison. (F) Flow cytometry of a rescue experiment using the indicated constructs, carried out as in (E). GFP-positive cells were analyzed: NPC1-/-, 2968 cells; NPC1 wild type, 2358 cells; P251/L929C/P249K/P259K, 3906 cells. Flow cytometry analyzed only GFP-positive cells of similar intensity.