Abstract
Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment. In this report, we use subcellular fractionation and microscopy to determine the localization of the murine Niemann-Pick C1 (NPC1) protein. Fractionation of mouse liver homogenates indicates that some NPC1 cosediments with lysosome-associated membrane protein 1 (LAMP1)-containing membranes. However, a significant amount of NPC1 is also found in membranes that do not contain LAMP1. Moreover, fractionation of liver membranes and fibroblasts in the presence of a nonionic detergent showed that a fraction of NPC1 cosediments with caveolin-1 in rafts. Immunofluorescence microscopy of cultured mouse fibroblasts showed that NPC1 is found in two morphologically distinct structures. The first population is characterized by large punctate structures that do not colocalize with major organelle protein markers, but do colocalize with filipin and a small fraction of caveolin-1. Examination of these large NPC1-containing compartments using electron microscopy shows that these structures contain extensive internal membranes. The second population is represented by smaller, more diffuse structures, a fraction of which colocalize with LAMP1-positive compartments. Incubation of fibroblasts with low density lipoprotein (LDL) increases colocalization of NPC1 with LAMP1-containing compartments. This colocalization can be further enhanced by treating fibroblasts with progesterone or chloroquine.The results indicate that NPC1 is associated with an unique vesicular compartment enriched with cholesterol and containing caveolin-1, and that NPC1 cycles to LAMP1-positive compartments, presumably to facilitate the processing of LDL-derived cholesterol.—Garver, W. S., R. A. Heidenreich, R. P. Erickson, M. A. Thomas, and J. M. Wilson. Localization of the murine Niemann-Pick C1 protein to two distinct intracellular compartments. J. Lipid Res. 2000. 41: 673–687.
Highlights
Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment
We demonstrate that after detergent extraction, Niemann-Pick C1 (NPC1) cosediments with caveolin-1 in detergent-insoluble fractions, suggesting that some NPC1 is associated with cholesterol and sphingomyelinenriched domains known as rafts [39]
Immunoblot analysis of cultured mouse fibroblasts and mouse liver homogenates derived from normal, NPC heterozygous, and NPC homozygous affected mice using the affinity-purified anti-NPC1 antiserum recognized a protein at 180 kD in normal cells that was not present in NPC homozygous affected cells (Fig. 1A)
Summary
Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment. The first population is characterized by large punctate structures that do not colocalize with major organelle protein markers, but do colocalize with filipin and a small fraction of caveolin-1 Examination of these large NPC1-containing compartments using electron microscopy shows that these structures contain extensive internal membranes. Incubation of fibroblasts with low density lipoprotein (LDL) increases colocalization of NPC1 with LAMP1-containing compartments. A smaller portion of lysosome-derived cholesterol is transported to the endoplasmic reticulum by a mechanism independent of the plasma membrane [19, 20]. For both pathways, the Golgi apparatus may have a role in the transport and tar-
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