The NPC1 family of proteins plays crucial roles in the intestinal absorption and intracellular trafficking of sterols. The Drosophila genome encodes two NPC1 homologs, one of which, NPC1a, is required for intracellular sterol trafficking in many tissues. Here we show that the other Drosophila NPC1 family member, NPC1b, is expressed in the midgut epithelium and that NPC1b is essential for growth during the early larval stages of development. NPC1b mutants are severely defective in sterol absorption, and the midgut epithelium of NPC1b mutants is deficient in sterols and sterol trafficking intermediates. By contrast, NPC1a mutants absorb sterols more efficiently than wild-type animals, and, unexpectedly, NPC1b;NPC1a double mutants absorb sterols as efficiently as wild-type animals. Together, these findings suggest that NPC1b plays an early role in sterol absorption, although sterol absorption continues at high efficiency through an NPC1a- and NPC1b-independent mechanism under conditions of impaired intracellular sterol trafficking.

The molecular mechanisms by which dietary cholesterol is trafficked within cells are poorly understood. Previous work indicates that the NPC1 family of proteins plays an important role in this process, although the precise functions performed by this protein family remain elusive. We have taken a genetic approach to further explore the NPC1 family in the fruit fly Drosophila melanogaster. The Drosophila genome encodes two NPC1 homologs, designated NPC1a and NPC1b, that exhibit 42% and 35% identity to the human NPC1 protein, respectively. Here we describe the results of mutational analysis of the NPC1a gene. The NPC1a gene is ubiquitously expressed, and a null allele of NPC1a confers early larval lethality. The recessive lethal phenotype of NPC1a mutants can be partially rescued on a diet of high cholesterol or one that includes the insect steroid hormone 20-hydroxyecdysone. We also find that expression of NPC1a in the ring gland is sufficient to rescue the lethality associated with the loss of NPC1a and that cholesterol levels in NPC1a mutant larvae are unchanged relative to controls. Our results suggest that NPC1a promotes efficient intracellular trafficking of sterols in many Drosophila tissues including the ring gland where sterols must be delivered to sites of ecdysone synthesis. S TEROLS are critical in eukaryotic biology as structural components of all membranes and as precursors for the synthesis of the large eukaryotic family of steroid hormones (Mouritsen and Zuckermann 2004;Payne and Hales 2004;Becher and McIlhinney 2005). While sterols are essential due to the important biological functions they serve, an overabundance of the primary mammalian sterol, cholesterol, is toxic and contributes to a variety of prevalent diseases including heart disease and stroke (Choy et al. 2004;Saini et al. 2004). Accumulating evidence also suggests that alterations in cholesterol homeostasis may contribute to neurodegenerative disorders such as Alzheimer's disease, although the mechanisms by which alterations in cholesterol metabolism affect neuronal integrity remain unclear (Burns and Duff 2002;Reiss et al. 2004;Wellington 2004).The importance of sterols in eukaryotic biology and disease pathogenesis has stimulated intensive investigation of cholesterol homeostasis in vertebrates. This body of work has clarified many features of cholesterol metabolism, including its de novo synthesis from acetate in the endoplasmic reticulum (ER) (Bloch 1965;Lynen 1966), its packaging into and transport within lipoprotein particles (Oncley 1954;Fredrickson 1957), and the receptor-mediated uptake of these particles by cells (Brown and Goldstein 1986). While these advances have fostered the development of treatment strategies that affect the etiology of hypercholesterolemia, this disorder remains a major source of morbidity, and our knowledge of cholesterol metabolism and the health consequences associated with altered cholesterol homeostasis is far from complete. In particular, the molecular mechanisms by which cholesterol is ab...