Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C–related protein reveals a primordial role in subcellular sphingolipid distribution

Malathi, Krishnamurthy; Higaki, Kazutaka; Tinkelenberg, Arthur H.; Balderes, Dina; Almanzar-Paramio, Dorca; Wilcox, Lisa J.; Erdeniz, Naz; Redican, Francis; Padamsee, Mahajabeen; Liu, Ying; Khan, Sohail; Alcantara, Frederick; Carstea, Eugene D.; Morris, Jill A.; Sturley, Stephen L.

doi:10.1083/jcb.200310046

Cited by 127 publications

(146 citation statements)

References 55 publications

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“…Given that the present data disrupted endocytic trafficking may only engage a small percentage of the total cholesterol pool. This is consistent with the yeast NPC model where altered localization of sphingolipids occurred without a large increase in ergosterol levels (61).…”

Section: Discussionsupporting

confidence: 77%

Accumulation of Glycosphingolipids in Niemann-Pick C Disease Disrupts Endosomal Transport

Vruchte

Lloyd-Evans

Veldman

et al. 2004

Journal of Biological Chemistry

168

138

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Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1 ؊/؊ mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.Increasing evidence suggests that the membranes of eukaryotic cells are not homogeneously fluid. They contain membrane microdomains, often referred to as lipid rafts and/or caveolae, enriched in glycosphingolipids (GSLs), 1 specific proteins, and cholesterol (1-3). Being operationally defined, i.e. by detergent insolubility and low buoyant density, the natural (in situ) existence of such membrane microdomains has been and continues to be controversial (4). However, they have recently been visualized by using electron microscopy as small structures covering as much as 35% of the cell surface (5). Although membrane microdomains on the cell surface tend to be transient (6), relatively stable microdomains have been visualized in the endocytic pathway (7) where they are proposed to play a role in protein and lipid sorting (8). Many GSL storage diseases, including Niemann-Pick type C (NPC), are often characterized by enlarged liver and spleen as well as severe neurodegeneration. In these diseases, cholesterol and GSLs accumulate in the endocytic pathway because of reduced breakdown. Hence, they may be a useful tool for determining the relative importance of GSLs in endocytic transport. Fibroblasts from patients suffering f...

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Section: Discussionsupporting

confidence: 77%

Accumulation of Glycosphingolipids in Niemann-Pick C Disease Disrupts Endosomal Transport

Vruchte

Lloyd-Evans

Veldman

et al. 2004

Journal of Biological Chemistry

168

138

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“…This contrasts with the itinerary for cholesterol in mammalian cells, in which lipoprotein-associated cholesterol is internalized by receptor-mediated endocytosis and is trafficked to late endosome/lysosomes. Third, deletion of Ncr1p in yeast indicates that this protein is not required for endocytic transport and sterol metabolism in yeast, because unlike in mammalian cells, such a deletion does not promote sterol accumulation in the vacuole (35,36). However, when Ncr1p is expressed in NPC1-null CHO cells, it is able to suppress the mutant phenotype, indicating that the yeast ortholog is competent with respect to sterol trafficking (35).…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, mutagenesis studies of the sterol-sensing domain of the NPC1 yeast ortholog, Ncr1p, have suggested a role for this region in cellular sphingolipid trafficking (35). In yeast, expression of the Y718D mutant, which corresponds to the mammalian SCAP D443N mutation, resulted in redistribution of PM-derived sphingolipids to the vacuole and other subcellular compartments, although no alterations in sterol metabolism were observed in this mutant.…”

Section: Discussionmentioning

confidence: 99%

“…Third, deletion of Ncr1p in yeast indicates that this protein is not required for endocytic transport and sterol metabolism in yeast, because unlike in mammalian cells, such a deletion does not promote sterol accumulation in the vacuole (35,36). However, when Ncr1p is expressed in NPC1-null CHO cells, it is able to suppress the mutant phenotype, indicating that the yeast ortholog is competent with respect to sterol trafficking (35). Whether expression of WT Ncr1p, which possesses a tyrosine at position 718 rather than the invariant aspartic acid residue found in the homologous regions of NPC1, SCAP, and HMG-CoA reductase, or the Ncr1p Y718D mutant would result in a sterolrelated gain of function phenotype in CHO cells remains to be determined.…”

Section: Discussionmentioning

confidence: 99%

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The Sterol-sensing Domain of the Niemann-Pick C1 (NPC1) Protein Regulates Trafficking of Low Density Lipoprotein Cholesterol

Millard

Gale

Dudley

et al. 2005

Journal of Biological Chemistry

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The Niemann-Pick C1 (NPC1) protein is a key participant in intracellular sterol trafficking and regulation of cholesterol homeostasis. NPC1 contains a pentahelical region that is evolutionarily related to sterol-sensing domains found in other polytopic proteins involved in sterol interactions or sterol metabolism, including sterol regulatory element-binding protein cleavage-activating protein and hydroxymethylglutaryl-CoA reductase. To gain insight into the role of the sterol-sensing domain of NPC1, we examined the effect of point mutations in the NPC1 sterol-sensing domain on the trafficking of low density lipoprotein-derived cholesterol and sphingolipids. We show that an NPC1 P692S loss of function mutation results in decreased cholesterol delivery to the plasma membrane and endoplasmic reticulum. By contrast, NPC1 proteins carrying a L657F or D787N point mutation, which correspond to the activating SCAP L315F and D443N mutations, respectively, exhibit a gain of function phenotype. Specifically, cell lines expressing the NPC1 L657F or D787N mutations show a nearly 2-fold increase in the rates of low density lipoprotein cholesterol trafficking to the plasma membrane and to the endoplasmic reticulum, and more rapid suppression of sterol regulatory element-binding protein-dependent gene expression. Trafficking of sphingolipids is intact in the D787N and L657F cell lines. Our finding that D787N and L657F are activating NPC1 mutations provide evidence for a conserved mechanism for the sterol-sensing domain among cholesterol homeostatic proteins.

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“…Cholesterol removal from the endosome depends on the NPC1 protein, which contains a sterol-sensing domain. Like its plasma membrane counterpart NPC1L1 [37 ], it transports cholesterol (and possibly sphingolipids [38]) towards the cytosol by a mechanism that is unclear but may involve vesiculation. Cholesterol also modulates vesicular traffic at and after the early endosomes via annexin II [39,40].…”

Section: Selective Endocytosismentioning

confidence: 99%

Membrane lipids and vesicular traffic

Meer

Sprong

2004

Current Opinion in Cell Biology

157

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Lipids were long considered to be passive passengers of carrier vesicles with the single role of sealing the transport container. We now know that specific phospholipids are required for efficient fusion, while others facilitate budding and fission. Moreover, the various polyphosphoinositides assist in the recruitment from the cytosol of proteins of the transport machinery. Finally, the segregation of membrane lipids into different fluid phases appears to serve as a 'lipid raft' mechanism for protein sorting at various stages of the secretory and endocytic pathways. The current challenge is to understand how proteins control the metabolism and subcellular localization, and thereby the activity, of the various lipids.

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Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C–related protein reveals a primordial role in subcellular sphingolipid distribution

Cited by 127 publications

References 55 publications

Accumulation of Glycosphingolipids in Niemann-Pick C Disease Disrupts Endosomal Transport

Accumulation of Glycosphingolipids in Niemann-Pick C Disease Disrupts Endosomal Transport

The Sterol-sensing Domain of the Niemann-Pick C1 (NPC1) Protein Regulates Trafficking of Low Density Lipoprotein Cholesterol

Membrane lipids and vesicular traffic

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