Mutations in the NPC1 gene cause Niemann-Pick type C disease, which is characterized by the accumulation of free cholesterol and other lipids in lysosomes. The NPC1 glycoprotein is located in a late endosomal compartment that transiently interacts with lysosomes. To identify factors regulating NPC1 expression and action, we analyzed the function of the human NPC1 promoter in human-derived ovarian, hepatic, and neuronal cells. A fragment containing the first 208 base pairs upstream from the major transcription initiation site was sufficient to drive near maximal NPC1 promoter activity. Deletion analysis revealed that sequences between base pairs -111 and -37 play an important role in controlling NPC1 transcription. Treatment of proliferating granulosa cells with 30 microM progesterone, which induces a reversible phenocopy of the cholesterol trafficking defect of Niemann-Pick type C disease, increased NPC1 mRNA levels threefold. The protein synthesis inhibitor, cycloheximide, also increased NPC1 mRNA levels, augmenting the progesterone-induced increase in NPC1 mRNA abundance. Progesterone treatment was shown to increase the mRNA half-life, but did not affect NPC1 promoter activity. Cysteine residues in a "cysteine-rich" loop predicted to reside in the intralumenal compartment of vesicles containing NPC1 were mutated, resulting in proteins that were incapable of correcting the cholesterol trafficking defect in CT60 cells, a Chinese hamster cell line in which the endogenous NPC1 gene is inactivated. Converting isoleucine 1061, also predicted to lie within the cysteine-rich loop, to a threonine residue inactivated the protein as well. The I1061T mutation is one of the most common mutations in Niemann-Pick type C disease. All of the cysteine-rich loop mutants were localized to cholesterol-engorged lysosomes in a pattern mimicking the distribution of NPC1 in progesterone-treated cells. A recombinant protein representing the cysteine-rich loop was shown to bind to a zinc-NTA agarose column. We conclude: (1) that cis elements residing in the first 111 base pairs upstream from the transcription start site are critical for transcription of the NPC1 gene; (2) that NPC1 expression is subject to posttranscriptional regulation in response to treatments that disrupt NPC1 function; and (3) that an intralumenal cysteine-rich loop with zinc-binding activity is critical to NPC1's ability to unload lysosomal cargo.
Copyright 2000 Academic Press.