eLife 4, e12177 (2016)

Cholesterol transport within cells is a well-understood process wherein low-density lipoprotein (LDL) is internalized via receptor-mediated endocytosis and trafficked to lysosomes, where cholesterol is ultimately liberated. Cholesterol is then transported by a lysosome-lumenal protein called NPC2 to the lysosomal membrane–embedded cholesterol efflux transporter NPC1. A deficiency of NPC1, as in the lysosomal storage disease Niemannn-Pick type C, leads to cholesterol accumulation within lysosomes. U18666A is a cationic amphiphile that inhibits the exit of LDL-derived cholesterol from lysosomes, mimicking NPC1 deficiency, though its target has not been formalized. To find the molecular target of U18666A, Lu et al. generated a photo-crosslinkable form of the compound, U-X, that also contained an alkyne group useful for a post-crosslinking attachment of functional elements such as fluorophores through click chemistry. Incubation of a CHO cell line with U-X identified a protein that was characterized as NPC1. The threshold for U18666A inhibition increased >100-fold with NPC1 overexpression, whereas NPC1 depletion resulted in a loss of U-X crosslinking. Testing several NPC1 point mutants that cause cholesterol transport deficiencies for their ability to crosslink with U-X led to the conclusion that U18666A binds to the sterol-sensing domain (SSD) of NPC1, not the cholesterol-binding N-terminal domain. These results suggest that the SSD contains a second cholesterol-binding site that may act in a relay to move cholesterol from lysosomes.