Mutations in the leucine zipper motif and sterol-sensing domain inactivatethe Niemann-Pick C1 glycoprotein

Niemann-Pick type C (NPC) disease, characterized by accumulation of low den sity lipoprotein-derived free cholesterol in lysosomes, is caused by mutati ons in the NPC1 gene. We examined the ability of wild-type NPC1 and NPC1 mu tants to correct the NPC sterol trafficking defect and their subcellular lo calization in CT60 cells. Cells transfected with wild-type NPC1 expressed 1 70- and 190-kDa proteins. Tunicamycin treatment resulted in a 140-kDa prote in, the deduced size of NPC1, suggesting that NPC1 is N-glycosylated, Mutat ion of all four asparagines in potential N-terminal N-glycosylation sites t o glutamines resulted in a 20-kDa reduction of the expressed protein. Prote ins with a single N-glycosylation site mutation localized to late endosome/ lysosomal compartments, as did wild-type NPC1, and each corrected the chole sterol trafficking defect. However, mutation of all four potential N-glycos ylation sites reduced ability to correct the NPC phenotype commensurate wit h reduced expression of the protein. Mutations in the putative sterol-sensi ng domain resulted in inactive proteins targeted to lysosomal membranes enc ircling cholesterol-laden cores. N-terminal leucine zipper motif mutants co uld not correct the NPC defect, although they accumulated in lysosomal memb ranes. We conclude that NPC1 is a glycoprotein that must have an intact ste rol-sensing domain and leucine zipper motif for cholesterol-mobilizing acti vity.