Niemann-Pick type C1 I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding

Over 200 disease-causing mutations have been identified in the NPC1 gene. The most prevalent mutation, NPC1^I1061T, is predicted to lie within the cysteine-rich luminal domain and is associated with the classic juvenile-onset phenotype of Niemann-Pick type C disease. To gain insight into the molecular mechanism by which the NPC1^I1061T mutation causes disease, we examined expression of the mutant protein in human fibroblasts homozygous for the NPC1^I1061T mutation. Despite similar NPC1 mRNA levels between wild type and NPC1^I1061T fibroblasts, NPC1 protein levels are decreased by 85% in NPC1^I1061T cells. Metabolic labeling studies demonstrate that unlike wild type protein, which undergoes a glycosylation pattern shift from Endo H-sensitive to Endo H-resistant species, NPC1^I1061T protein remains almost exclusively Endo H-sensitive and exhibits a reduced half-life (t_1?2 6.5 h) versus wild type Endo H-resistant species (t_1?2 42 h). Treatment with chemical chaperones, growth at permissive temperature, or inhibition of proteasomal degradation increases NPC1^I1061T protein levels, indicating that the mutant protein is likely targeted for endoplasmic reticulum-associated degradation (ERAD) due to protein misfolding. Overexpression of NPC1^I1061T in NPC1-deficient cells results in late endosomal localization of the mutant protein and complementation of the NPC mutant phenotype, likely due to a small proportion of the nascent NPC1^I1061T protein that is able to fold correctly and escape the endoplasmic reticulum quality control checkpoints. Our findings provide the first description of an endoplasmic reticulum trafficking defect as a mechanism for human NPC disease, shedding light on the mechanism by which the NPC1^I1061T mutation causes disease and suggesting novel approaches to treat NPC disease caused by the NPC1^I1061T mutation.