Saccharomyces cerevisiae Npc2p Is a Functionally Conserved Homologue of the Human Niemann-Pick Disease Type C 2 Protein, hNPC2

Abstract

Niemann-Pick Disease Type C (NP-C) is a fatal neurodegenerative disease, which is biochemically distinguished by the lysosomal accumulation of exogenously derived cholesterol. Mutation of either the hNPC1 or hNPC2 gene is causative for NP-C. We report the identification of the yeast homologue of human NPC2, Saccharomyces cerevisiae Npc2p. We demonstrate that scNpc2p is evolutionarily related to the mammalian NPC2 family of proteins. We also show, through colocalization, subcellular fractionation, and secretion analyses, that yeast Npc2p is treated similarly to human NPC2 when expressed in mammalian cells. Importantly, we show that yeast Npc2p can efficiently revert the unesterified cholesterol and GM1 accumulation seen in hNPC2 ؊/؊ patient fibroblasts demonstrating that it is a functional homologue of human NPC2. The present study reveals that the fundamental process of NPC2-mediated lipid transport has been maintained throughout evolution.Niemann-Pick Disease Type C (NP-C) is a fatal neurovisceral disorder that is characterized by the accumulation of low-density-lipoprotein (LDL)-derived cholesterol in endolysosomal compartments (37). NP-C is caused by mutation of either of two genes: hNPC1 (5, 30) or hNPC2 (34). Mutations in hNPC1 account for 95% of all patients (6, 37). Recently, it was demonstrated that the yeast homologue of hNPC1, scNcr1p, can functionally complement the loss of hNPC1 from mutant CHO cells (31). This finding suggests that there is an evolutionary conservation of function between species.Human NPC2 (hNPC2), which is mutated in approximately 5% of NP-C disease (37), encodes a conserved 151-amino-acid secreted glycoprotein with an endoplasmic reticulum signal sequence (24). The NPC2 protein contains an MD-2-related lipid-recognition (ML) domain (21), which is predicted to mediate direct binding to lipids (21). This prediction is based in part on the finding that the MD-2 protein, the founding member of the ML domain family of proteins, binds directly to lipopolysaccharide (49). Structural analysis of an ML domaincontaining protein, the dust mite allergen Der P 2, reveals that this protein folds into two ␤-strands with an internal cavity that is occupied by a hydrophobic ligand predicted to be a lipid (10). As seen for the other ML domain-containing proteins tested thus far (10, 49), the human, mouse, and porcine NPC2 orthologues all bind directly to lipid ligands, specifically cholesterol or cholesterol analogs (13,26,35). The recently solved crystal structure of bovine NPC2 (bNPC2) is consistent with a model wherein cholesterol binds within a loosely packed hydrophobic protein core (13). Taken together, these binding and structural studies suggest that NPC2 plays a direct role in sterol transport.Here we report the identification of the yeast homologue of hNPC2 that we have termed Npc2p (scNpc2p). Our analysis of Saccharomyces cerevisiae Npc2p through phylogenetic and homology modeling studies suggests that this protein has been conserved from yeast to mammals. Most importantly, we show that...

An “Exacerbate-reverse” Strategy in Yeast Identifies Histone Deacetylase Inhibition as a Correction for Cholesterol and Sphingolipid Transport Defects in Human Niemann-Pick Type C Disease

“…The mammalian and yeast NPC orthologs are interchangeable to the extent that the yeast orthologs of NPC1 and NPC2 complement lesions in the corresponding genes in cultured mammalian cells (7,8). There is 35% amino acid sequence identity between the human and yeast NPC1 orthologs, and both proteins localize to the limiting membranes of the lysosomal (vacuolar)/endosomal systems.…”

Drosophila Niemann-Pick Type C-2genes control sterol homeostasis and steroid biosynthesis: a model of human neurodegenerative disease

“…To uncover the disease mechanisms as well as the biological function(s) of NPC proteins, useful NPC disease models have been established in yeast, worms, flies and mice (Berger et al, 2005;Higaki et al, 2004;Li et al, 2004;Malathi et al, 2004). We and the L. Pallanck laboratory have previously created Drosophila NPC models using npc1a (also referred to as NPC1 -FlyBase) mutations (Fluegel et al, 2006;Huang et al, 2005).…”

Niemann–Pick Type C Disease Proteins: Orphan Transporters Or Membrane Rheostats?

“…Thus, much of our insight on protein function stems from analyses of orthologs of NPC1 (Table 1) and NPC2 in model systems. This approach is validated by complementation studies of the yeast orthologs to NPC1 and NPC2 in mammalian cells [12,29], and further by reports that 43% of missense mutations and 15% of missense polymorphisms in NPC1 in human NPC patients are at phylogenically conserved residues [15]. Together, these data suggest an evolutionarily conserved function is disturbed in NPC patients.…”

“…Expression of the yeast genes corrects the lipid trafficking defect of NPC1 −/− CHO cells or human NPC2 −/− patient fibroblasts, respectively [12,29]. However, sterol imbalance was not associated with loss of these pathways in yeast [12,35,36].…”