In contrast to lipoprotein-mediated sterol uptake, free sterol influx by eukaryotic cells is poorly understood. To identify components of non-lipoprotein-mediated sterol uptake, we utilized strains of Saccharomyces cerevisiae that accumulate exogenous sterol due to a neomorphic mutation in the transcription factor, UPC2. Two congenic upc2-1 strains, differing quantitatively in aerobic sterol uptake due to a modifying mutation in the HAP1 transcription factor, were compared using DNA microarrays. We identified 9 genes as responsive to UPC2 that were also induced under anaerobiosis, when sterol uptake is essential. Deletion mutants in these genes were assessed for sterol influx in the upc2-1 background. UPC2 itself was up-regulated under these conditions and was required for aerobic sterol influx. Deletion of the ATP-binding cassette transporters YOR011w (AUS1) or PDR11, or a putative cell wall protein encoded by DAN1, significantly reduced sterol influx. Sodium azide and vanadate inhibited sterol uptake, consistent with the participation of ATP-binding cassette transporters. We hypothesized that the physiological role of Aus1p and Pdr11p is to mediate sterol uptake when sterol biosynthesis is compromised. Accordingly, expression of AUS1 or PDR11 was required for anaerobic growth and sterol uptake. We proposed similar molecules may be important components of sterol uptake in all eukaryotes.

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The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases. Four of these genes are similar to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1 yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor. The residual triglyceride biosynthesis that persists in the LRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol acyltransferase. These mechanisms may also exist in mammalian cells.Triglyceride (TG) 1 biosynthesis is a common method of energy storage and thus has an important role in energy balance. In humans, overaccumulation of TG, either as obesity or elevated serum triglyceride, has been shown to be an independent risk factor for a variety of diseases including diabetes (1) and atherosclerosis (2, 3). In the pathway described by Kennedy (4) for glyceride and glycerophosphatide synthesis, a branch point is reached at diacylglycerol (DG) that can serve as a precursor for several phospholipid species and as a substrate for acylCoA, diacylglycerol O-acyltransferase (DGAT) (EC 2.3.1.20), which catalyzes the terminal step in TG synthesis. Expression of a recently identified mammalian DGAT cDNA in insect and mammalian cells conferred elevated triglyceride synthesis but did not change incorporation of fatty acids into sterol ester (5-7). The DGAT gene belongs to the acyl-CoA cholesterol acyltransferase (ACAT) gene family that includes two mammalian ACATs (ACAT1, ACAT2) and two yeast ACAT-related enzymes (ARE1, ARE2) that catalyze intracellular sterol esterification (8). Whereas yeast can synthesize TG from oleoyl-CoA and DG, deletion mutants in ARE1 and ARE2 do not reduce [ 3 H]oleate incorporation into TG (9). Therefore, a conspicuous absence from the ACAT gene family is a yeast DGAT.Further examination of the Saccharomyces cerevisiae genome data base revealed two DGAT-like genes in addition to the ARE genes. We show here that these four genes do not have a major role in TG synthesis in yeast. By contrast, a yeast gene with sequence similarity to mammalian lecithin-cholesterol acyltransferase (LCAT) (EC 2.3.1.43) catalyzes the esterification of DG using phosphatidylcholine as the acyl donor. This novel enzymatic reaction mediates the majority of TG synthesis in the yeast cell during exponential growth. EXPERIMENTAL PROCEDURESGeneral-Molecular biology a...

Lipid movement between organelles is a critical component of eukaryotic membrane homeostasis. Niemann Pick type C (NP-C) disease is a fatal neurodegenerative disorder typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of yeast NP-C–related gene 1 (NCR1), the orthologue of the human NP-C gene 1 (NPC1) defective in the disease, in Chinese hamster ovary NPC1 mutant cells suppressed lipid accumulation. Deletion of NCR1, encoding a transmembrane glycoprotein predominantly residing in the vacuole of normal yeast, gave no phenotype. However, a dominant mutation in the putative sterol-sensing domain of Ncr1p conferred temperature and polyene antibiotic sensitivity without changes in sterol metabolism. Instead, the mutant cells were resistant to inhibitors of sphingolipid biosynthesis and super sensitive to sphingosine and C2-ceramide. Moreover, plasma membrane sphingolipids accumulated and redistributed to the vacuole and other subcellular membranes of the mutant cells. We propose that the primordial function of these proteins is to recycle sphingolipids and that defects in this process in higher eukaryotes secondarily result in cholesterol accumulation.

Intracellular cholesterol redistribution between membranes and its subsequent esterification are critical aspects of lipid homeostasis that prevent free sterol toxicity. To identify genes that mediate sterol trafficking, we screened for yeast mutants that were inviable in the absence of sterol esterification. Mutations in the novel gene, ARV1, render cells dependent on sterol esterification for growth, nystatin-sensitive, temperaturesensitive, and anaerobically inviable. Cells lacking Arv1p display altered intracellular sterol distribution and are defective in sterol uptake, consistent with a role for Arv1p in trafficking sterol into the plasma membrane. Human ARV1, a predicted sequence ortholog of yeast ARV1, complements the defects associated with deletion of the yeast gene. The genes are predicted to encode transmembrane proteins with potential zincbinding motifs. We propose that ARV1 is a novel mediator of eukaryotic sterol homeostasis.Sterols are essential structural and regulatory components of eukaryotic cellular membranes (1, 2). However, cholesterol over-accumulation is cytotoxic (3), necessitating mechanisms to maintain this metabolite at appropriate levels. A pivotal component of this homeostasis is the esterification of free sterol by acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) 1 (4, 5).Indeed, the inhibition of ACAT in sterol-loaded cells induces cell death when extracellular sterol acceptors such as high density lipoproteins are absent (6, 7). Intracellular cholesterol redistribution mediates a number of responses to elevated free sterol levels. These include elevated ACAT activity, down-regulated sterol and fatty acid biosynthesis, and reduced lipoprotein uptake via LDL receptors (8, 9). The latter two events reflect changes in transcriptional activation by sterol regulatory element-binding proteins (SREBPs) in response to sterol accumulation in regulatory pools (9), whereas ACAT activity is allosterically regulated by substrate supply (10). Sterols are maintained at a high concentration in the plasma membrane (PM) relative to the endoplasmic reticulum (ER) (1, 2), where SREBP and ACAT reside. Thus trafficking of sterol to and from the ER is a critical component of sterol homeostasis.The process of sterol trafficking is poorly understood at the molecular level. In certain cell types, caveolin influences what has been termed "fast" movement of cholesterol to plasma membrane cholesterol-rich microdomains (caveolae) (11,12). Mutations in the Niemann Pick type C (NPC1) gene result in accumulation of LDL-derived cholesterol in the lysosome (13, 14). However, not all cells express caveolin, and the movement of endogenously synthesized cholesterol to the plasma membrane in NPC1-deficient cells is normal (15).To identify novel genes that mediate sterol trafficking in all higher cells, we utilized the genetically tractable model eukaryote, Saccharomyces cerevisiae (budding yeast). We reasoned that dependence on sterol esterification for viability would be one criterion for identifying novel sterol...