NPC1L1, a recently identified relative of NiemannPick C1, was characterized to determine its subcellular location and potential function(s). NPC1L1 was highly expressed in HepG2 cells and localized in a subcellular vesicular compartment rich in the small GTPase Rab5. mRNA expression profiling revealed significant differences between mouse and man with highest expression found in human liver and significant expression in the small intestine. In contrast, liver expression in mouse was extremely low with mouse small intestine exhibiting the highest NPC1L1 expression. A mouse knock-out model of NPC1L1 was generated and revealed that mice lacking a functional NPC1L1 have multiple lipid transport defects. Surprisingly, lack of NPC1L1 exerts a protective effect against diet-induced hyperlipidemia. Further characterization of cell lines generated from wildtype and knock-out mice revealed that in contrast to wild-type cells, NPC1L1 cells exhibit aberrant plasma membrane uptake and subsequent transport of various lipids, including cholesterol and sphingolipids. Furthermore, lack of NPC1L1 activity causes a deregulation of caveolin transport and localization, suggesting that the observed lipid transport defects may be the indirect result of an inability of NPC1L1 null cells to properly target and/or regulate caveolin expression.Niemann Pick C1-like 1 protein (NPC1L1) 1 was previously identified based on its high degree of similarity, 42% amino acid identity and 51% similarity to the polytopic, late endosome-resident protein, NPC1. Both possess a putative sterolsensing domain, suggesting roles in sterol/lipid transport (1), and they also have an amino-terminal "NPC1 domain" (2). Based on this homology and preliminary data, we hypothesized that NPC1L1 has a lipid permease function similar to that of NPC1 (3). In contrast, however, the two proteins have variant targeting signals and thus are predicted to function similarly but at different intracellular locations.To gain a further understanding of the function of this family of proteins, we have carried out cell and molecular studies to determine the location and tissue expression of NPC1L1. In addition, since no known disorders map at 7p13, the chromosomal location of human NPC1L1 (1), we have generated a mouse knock-out of NPC1L1. Our results are in contrast to a published report suggesting that NPC1L1 resides at the plasma membrane (4) and indicate that this protein is predominantly intracellular and colocalizes with the small GTPase Rab5. In addition, the expression profile of human NPC1L1 shows this protein to be highly enriched in liver. Finally, inactivation of NPC1L1 leads to multiple lipid transport defects including cholesterol and sphingolipids, suggesting that NPC1L1 plays a critical role in lipid homeostasis and transport in support of our original hypothesis.
MATERIALS AND METHODSTissue Culture, Transfection, and Immunofluorescence Studies-All cells, including COS7, HepG2, and Caco-2 cells, were obtained from ATCC (Manassas, VA). Cells were maintained at 37°C in...