Abstract
Mutations in the late endosomal/lysosomal membrane protein Niemann-Pick C1 (NPC1) are known to cause a generalized block in retrograde vesicle-mediated transport, resulting in the hyper-accumulation of multiple lysosomal cargos. An important, yet often overlooked, category of lysosomal cargo includes the vast array of small molecular weight amine-containing molecules that are substrates for ion trapping in the highly acidic organelle lumen. We show here that the introduction of amine-containing molecules in lysosomes can significantly stimulate NPC1-mediated late endosome/lysosome fusion, and subsequently the secretion of lysosomal cargo. To illustrate the physiological importance of this NPC1-mediated transport pathway, we show that NPC1-deficient cells are more susceptible to the toxic effects of a lysosomotropic polyamine metabolite 3-aminopropanal. Moreover, NPC fibroblasts are shown to have higher levels of polyamine oxidase, an enzyme involved in the formation of 3-aminopropanal. Collectively, these findings provide strong support for a novel functional role for NPC1 and may also provide clues toward understanding NPC disease progression.
Highlights
24584 JOURNAL OF BIOLOGICAL CHEMISTRY other lipids have been quite effective in reducing the hyperaccumulation phenotype; such approaches have not been successful in significantly decreasing Niemann-Pick type C (NPC) disease progression in various human and animal disease models [5,6,7,8]
There are three separate lines of evidence that support a potential functional role for Niemann-Pick C1 (NPC1) in the regulation of endogenous amines in lysosomes: 1) We and others have shown that NPC1 is required for efficient clearance of amines from lysosomes [23, 24]; 2) endogenous hydrophobic amines have been shown to be over 20-fold elevated in livers of mice with NPC disease compared with control mice and were shown to be con
Cells deficient in NPC1 are shown to be highly sensitive to the toxic effects of an endogenous polyamine metabolite, which illustrates the significance of the function
Summary
Antibodies and Reagents—Anti-EEA1 (early endosomes), anti-golgin-84, and anti-GM130 (Golgi apparatus) mouse monoclonal antibodies were from BD Biosciences (San Jose, CA). For siRNA experiments requiring subsequent amine exposure, transfections were performed as described, at which point cells were exposed to 70 M NR or 100 M chloroquine for 6 h. Cells were subsequently washed with PBS (3ϫ), and secondary antibodies (Alexa Fluor 647-conjugated anti-mouse or rabbit IgG, 1:1000) were applied for 2 h. The isolation procedure, as well as control experiments, was performed as previously described with minor modifications to allow for localization of the superparamagnetic iron dextran in lysosomes of amine-treated cells [30]. To confirm that the iron dextran was localizing in aminecontaining compartments, cells were incubated with 2 mg/ml Alexa Fluor 647 dextran in PBS for 2 h, washed with PBS (4ϫ), and subsequently given chase in complete dextran-free medium for 20 h at 37 °C. Fractions of purified cytosol were assayed for -hexosaminidase activity, a lysosomal enzyme, as described by Duvvuri et al [19]
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