Targeting of NPC1 to Late Endosomes Involves Multiple Signals, Including One Residing within the Putative Sterol-sensing Domain

Scott, Catherine; Higgins, Maureen E.; Davies, Joanna P.; Ioannou, Yiannis A.

doi:10.1074/jbc.m406090200

Cited by 36 publications

(32 citation statements)

References 57 publications

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“…While these observations complicate efforts to define strict orthologous relationships within this gene family, several sequence features suggest that NPC1a and NPC1b may function equivalently to NPC1 and NPC1L1, respectively. In particular, the findings that NPC1a has greater sequence similarity to NPC1 and that NPC1a and NPC1 (but not NPC1b or NPC1L1) share a C-terminal lysosomal targeting motif suggest that NPC1a may play a functional role that more closely parallels that of NPC1 (Ioannou 2000;Scott et al 2004). Furthermore, the NPC1L1 and NPC1b polypeptides share a putative YQRL Golgi retention signal (Humphrey et al 1993) and no such sequence is found in NPC1 or NPC1a (see supplemental Figure 1 at http://www.genetics.org/supplemental/).…”

Section: Resultsmentioning

confidence: 99%

“…The NPC1 protein also bears resemblance to the bacterial class of RND permeases and is able to transport fatty acids (Davies et al 2000a), although the importance of this result is unclear as the flux of these species from lysosomes in NPCD cells is unperturbed (Passeggio and Liscum 2005). Consistent with the predicted topology and lysosomal targeting motifs present in NPC1, subcellular distribution analyses indicate that the NPC1 polypeptide is localized to membranes of late endosomes, lysosomes, and the TGN (Garver et al 2000;Scott et al 2004). In conjunction with the cholesterol accumulation phenotype seen upon loss of NPC1 function, these findings strongly suggest a direct role for this factor in the trafficking of sterols through the endocytic/secretory pathway, although precisely how NPC1 mediates these trafficking events is far from clear.…”

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confidence: 98%

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Mutations of a Drosophila NPC1 Gene Confer Sterol and Ecdysone Metabolic Defects

2006

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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...

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Section: Resultsmentioning

confidence: 99%

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confidence: 98%

Mutations of a Drosophila NPC1 Gene Confer Sterol and Ecdysone Metabolic Defects

2006

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“…NPC1 protein is predominantly located within the late endosomal membrane but is also transiently associated with lysosomes and the trans-Golgi network (10). Multiple peptide sequences within the protein are responsible for targeting to the endosomal compartment (11). Late endosomes are comprised of limiting membranes and internal membranes (12).…”

Section: Biochemical Studies On Npc1 and Npc2 Proteinsmentioning

confidence: 99%

Niemann-Pick Type C Disease and Intracellular Cholesterol Trafficking

Chang

Reid

Sugii

et al. 2005

Journal of Biological Chemistry

150

107

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“…2A). To examine whether endosomal localization was required for NPC1 to undergo this modification, we examined effects of cholesterol depletion on the ␦LLNF mutant protein that lacks the C-terminal di-leucine motif and is retained in the ER (Watari et al, 1999b;Scott et al, 2004). As in the case of the P691S mutant, ubiquitylation of this mutant protein was barely affected by cholesterol depletion (Fig.…”

Section: Mutant Proteins Flag-npc1(p691s) and Flag-npc1(␦llnf) Failedmentioning

confidence: 99%

Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4

Ohsaki

Sugimoto

Suzuki

et al. 2006

Journal of Cell Science

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Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain, whereas NPC2 is a soluble protein that contains an MD-2-like lipid-recognition domain. In the current study, we addressed the hypothesis that ubiquitylation of NPC1 might be regulated by cholesterol. We found that depletion of cellular cholesterol facilitated ubiquitylation of NPC1 expressed in COS cells. A loss-of-function mutant, NPC1(P691S), which contains an amino acid substitution in the sterol-sensing domain, failed to respond to cholesterol depletion. Another mutant, NPC1(δLLNF), which lacks the endosomal-targeting motif, also failed to respond. SKD1(E235Q), a dominant-negative mutant of SKD1/Vps4 that inhibits disassembly of the endosomal sorting complex required for transport (ESCRT), caused an accumulation of ubiquitylated NPC1. SKD1(E235Q) associated with NPC1 on the endosomal membrane, whereas wild-type SKD1 associated with NPC1 only when cells were depleted of cholesterol. Similarly, in control human skin fibroblasts, cholesterol depletion facilitated ubiquitylation of endogenous NPC1. In patient cells that lack NPC2 function, NPC1 was ubiquitylated regardless of cellular cholesterol levels, suggesting that NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions. These results suggest that ubiquitylation of NPC1 and its association with the ESCRT complex are controlled by endosomal cholesterol levels utilizing a mechanism that involves NPC2.

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Targeting of NPC1 to Late Endosomes Involves Multiple Signals, Including One Residing within the Putative Sterol-sensing Domain

Cited by 36 publications

References 57 publications

Mutations of a Drosophila NPC1 Gene Confer Sterol and Ecdysone Metabolic Defects

Mutations of a Drosophila NPC1 Gene Confer Sterol and Ecdysone Metabolic Defects

Niemann-Pick Type C Disease and Intracellular Cholesterol Trafficking

Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4

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