Showing papers on "Sterol published in 2005"
TL;DR: A model is proposed in which sterol and membrane binding promote reciprocal conformational changes that facilitate a sterol transfer and signalling cycle and shows that sterol binding closes the lid and stabilizes a conformation favouring transport across aqueous barriers and signal transmission.
Abstract: The oxysterol-binding-protein (OSBP)-related proteins (ORPs) are conserved from yeast to humans1,2, and are implicated in the regulation of sterol homeostasis3,4 and in signal transduction pathways5. Here we report the structure of the full-length yeast ORP Osh4 (also known as Kes1) at 1.5–1.9 A resolution in complexes with ergosterol, cholesterol, and 7-, 20- and 25-hydroxycholesterol. We find that a single sterol molecule binds within a hydrophobic tunnel in a manner consistent with a transport function for ORPs. The entrance is blocked by a flexible amino-terminal lid and surrounded by basic residues that are critical for Osh4 function. The structure of the open state of a lid-truncated form of Osh4 was determined at 2.5 A resolution. Structural analysis and limited proteolysis show that sterol binding closes the lid and stabilizes a conformation favouring transport across aqueous barriers and signal transmission. The structure of Osh4 in the absence of ligand exposes potential phospholipid-binding sites that are positioned for membrane docking and sterol exchange. On the basis of these observations, we propose a model in which sterol and membrane binding promote reciprocal conformational changes that facilitate a sterol transfer and signalling cycle.
399 citations
TL;DR: It has become clear that the cyclases that produce hopanoids from squalene and sterols and pentacyclic triterpenoids from oxidosqualene (OSCs) are closely related and reflect evolutionary changes in this biosynthetic pathway over time.
316 citations
TL;DR: RCT and cholesterol efflux play a major role in anti-atherogenesis, and modification of these processes may provide new therapeutic approaches to cardiovascular disease.
Abstract: Reverse cholesterol transport (RCT) is a pathway by which accumulated cholesterol is transported from the vessel wall to the liver for excretion, thus preventing atherosclerosis. Major constituents of RCT include acceptors such as high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I), and enzymes such as lecithin:cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), hepatic lipase (HL) and cholesterol ester transfer protein (CETP). A critical part of RCT is cholesterol efflux, in which accumulated cholesterol is removed from macrophages in the subintima of the vessel wall by ATP-binding membrane cassette transporter A1 (ABCA1) or by other mechanisms, including passive diffusion, scavenger receptor B1 (SR-B1), caveolins and sterol 27-hydroxylase, and collected by HDL and apoA-I. Esterified cholesterol in the HDL is then delivered to the liver for excretion. In patients with mutated ABCA1 genes, RCT and cholesterol efflux are impaired and atherosclerosis is increased. In studies with transgenic mice, disruption of ABCA1 genes can induce atherosclerosis. Levels of HDL are inversely correlated with incidences of cardiovascular disease. Supplementation with HDL or apoA-I can reverse atherosclerosis by accelerating RCT and cholesterol efflux. On the other hand, pro-inflammatory factors such as interferon-gamma (IFN-gamma), endotoxin, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), can be atherogenic by impairing RCT and cholesterol efflux, according to in vitro studies. RCT and cholesterol efflux play a major role in anti-atherogenesis, and modification of these processes may provide new therapeutic approaches to cardiovascular disease. Further research on new modifying factors for RCT and cholesterol efflux is warranted.
307 citations
TL;DR: A model in which Sre1 and Scp1 monitor oxygen-dependent sterol synthesis as an indirect measure of oxygen supply and mediate a hypoxic response in fission yeast is proposed and tested.
301 citations
TL;DR: At recommended intakes of about 2 to 2.5 g/day, products enriched with plant stanol/sterol esters lower plasma LDL cholesterol levels by 10% to 14% without any reported side effects, and plant stanols/sterols can be considered to be effective and safe cholesterol-lowering functional food ingredients.
Abstract: Incorporation of plant stanol esters into margarine is among the first examples of a functional food with proven low-density lipoprotein (LDL) cholesterol-lowering effectiveness. Recently, there have been many studies on the effects of plant stanols/sterols on cholesterol metabolism. It has been found that the serum LDL cholesterol-lowering effect of plant stanols/sterols originates from reduced intestinal cholesterol absorption, a process in which changes in micellar composition are thought to play a major role. However, recent findings suggest that there is an additional process in which plant stanols/sterols actively influence cellular cholesterol metabolism within intestinal enterocytes. Furthermore, in response to the reduced supply of exogenous cholesterol, receptor-mediated lipoprotein cholesterol uptake is probably enhanced, as shown by increased LDL receptor expression. At recommended intakes of about 2 to 2.5 g/day, products enriched with plant stanol/sterol esters lower plasma LDL cholesterol levels by 10% to 14% without any reported side effects. Thus, plant stanols/sterols can be considered to be effective and safe cholesterol-lowering functional food ingredients.
294 citations
TL;DR: It is concluded that the cholesterol biosynthetic pathway is impaired in HD cells, mice, and human subjects, and that the search for HD therapies should also consider cholesterol levels as both a potential target and disease biomarker.
Abstract: The expansion of a polyglutamine tract in the ubiquitously expressed huntingtin protein causes Huntington's disease (HD), a dominantly inherited neurodegenerative disease. We show that the activity of the cholesterol biosynthetic pathway is altered in HD. In particular, the transcription of key genes of the cholesterol biosynthetic pathway is severely affected in vivo in brain tissue from HD mice and in human postmortem striatal and cortical tissue; this molecular dysfunction is biologically relevant because cholesterol biosynthesis is reduced in cultured human HD cells, and total cholesterol mass is significantly decreased in the CNS of HD mice and in brain-derived ST14A cells in which the expression of mutant huntingtin has been turned on. The transcription of the genes of the cholesterol biosynthetic pathway is regulated via the activity of sterol regulatory element-binding proteins (SREBPs), and we found an approximately 50% reduction in the amount of the active nuclear form of SREBP in HD cells and mouse brain tissue. As a consequence, mutant huntingtin reduces the transactivation of an SRE-luciferase construct even under conditions of SREBP overexpression or in the presence of an exogenous N-terminal active form of SREBP. Finally, the addition of exogenous cholesterol to striatal neurons expressing mutant huntingtin prevents their death in a dose-dependent manner. We conclude that the cholesterol biosynthetic pathway is impaired in HD cells, mice, and human subjects, and that the search for HD therapies should also consider cholesterol levels as both a potential target and disease biomarker.
268 citations
TL;DR: It is shown that lanosterol, the first sterol intermediate in cholesterol synthesis, potently stimulates ubiquitination of reductase, whereas cholesterol has no effect at 10-fold higher concentrations, and a pair of methyl groups located in the C4 position of Lanosterol confers this differential response.
260 citations
TL;DR: The mechanism by which newly synthesized sterols are transported from their site of synthesis, the endoplasmic reticulum (ER), to the sterol-enriched plasma membrane (PM) is not fully understood and it is suggested that transport occurs by equilibration of ER and PM ergosterol pools via a bidirectional, nonvesicular process that is saturated in wild-type exponentially growing yeast.
Abstract: The mechanism by which newly synthesized sterols are transported from their site of synthesis, the endoplasmic reticulum (ER), to the sterol-enriched plasma membrane (PM) is not fully understood. Studies in mammalian cells suggest that newly synthesized cholesterol is transported to the PM in Golgi-bypassing vesicles and/or via a nonvesicular process. Using the yeast Saccharomyces cerevisiae as a model system, we now rule out an essential role for known vesicular transport pathways in transporting the major yeast sterol, ergosterol, from its site of synthesis to the PM. We use a cyclodextrin-based sterol capture assay to show that transport of newly synthesized ergosterol to the PM is unaltered in cells defective in Sec18p, a protein required for almost all intracellular vesicular trafficking events; we also show that transport is not blocked in cells that are defective in formation of transport vesicles at the ER or in vesicle fusion with the PM. Our data suggest instead that transport occurs by equilibration (t(1/2) approximately 10-15 min) of ER and PM ergosterol pools via a bidirectional, nonvesicular process that is saturated in wild-type exponentially growing yeast. To reconcile an equilibration process with the high ergosterol concentration of the PM relative to ER, we note that a large fraction of PM ergosterol is found condensed with sphingolipids in membrane rafts that coexist with free sterol. We propose that the concentration of free sterol is similar in the PM and ER and that only free (nonraft) sterol molecules have access to a nonvesicular transport pathway that connects the two organelles. This is the first description of biosynthetic sterol transport in yeast.
211 citations
TL;DR: The ability to manipulate the mass of maternal cholesterol that crosses to the fetus could result in a dramatic improvement in the development of fetuses that lack the ability to synthesize cholesterol, such as those with Smith-Lemli-Opitz syndrome.
187 citations
TL;DR: In this article, the effects of PPARdelta activation by GW610742 were evaluated in wild-type and Abca1-deficient (Abca1(-/-)) mice that lack HDL.
181 citations
TL;DR: An important part of excess cholesterol is excreted directly via the intestine, supporting the existence of an alternative, quantitatively important route for cholesterol disposal.
TL;DR: Values for the composition of the sterols, triacylglycerols, fatty acids, iodine value, and tocopherol composition were generally in good agreement with the results of previous similar surveys.
Abstract: This paper describes the composition of walnut oils obtained from nuts collected from seven countries that are major suppliers of walnut oil. Oils were extracted from the nuts using small-scale industry pressing equipment and analyzed using standard methods for fatty acids, fatty acids in the triacylglycerol 2-position, tocopherols and tocotrienols, triacylglycerols, sterols, steradienes, and iodine value. Values for the composition of the sterols, triacylglycerols, fatty acids, iodine value, and tocopherol composition were generally in good agreement with the results of previous similar surveys. Tocotrienols were not detected in any sample. Steradienes (stigmastadiene, campestadiene, stigmastatriene, and campestatriene) were not detected in any oil.
TL;DR: These new findings contribute to the understanding of cholesterol and plant sterol concentrations in serum, and the effect of dietary and drug intervention to reduce serum concentrations of sterols.
Abstract: The recent discovery of transporters in the intestinal mucosa and the canalicular membrane has given new insights into the regulation of intestinal absorption as well as the biliary output of cholesterol and plant sterols. The 2 adenosine triphosphate (ATP)-binding cassette (ABC) half-transporters ABCG5 and ABCG8 are expressed in the mucosa cells and the canalicular membrane, and they resecrete sterols, especially absorbed plant sterols, back into the intestinal lumen and from the liver into bile. Defects of either of these cotransporters lead to the rare inherited disease of phytosterolemia, which is clinically defined by hyperabsorption and diminished biliary excretion of plant sterols. Furthermore, it has been recently demonstrated that the Niemann-Pick C1-Like 1 (NPC1L1) transporter is most likely responsible for the transport of cholesterol and plant sterols from the brush border membrane into the intestinal mucosa. Ezetimibe interferes with NPC1L1, reducing the intestinal uptake of cholesterol and plant sterols. These new findings contribute to our understanding of cholesterol and plant sterol concentrations in serum, and the effect of dietary and drug intervention to reduce serum concentrations of sterols.
TL;DR: The data support the idea that ergosterol and cholesterol do enhance survivability for cells exposed to high concentrations of ethanol and provide evidence that the appearance of the interdigitated (L(beta)I) phase bilayer is a major factor in the disruption of cellular activity.
TL;DR: Direct evidence is provided that lipid peroxidation alters the essential organization and structure of membrane lipids in a manner that may contribute to changes in membrane function during aging and oxidative stress-related disorders.
TL;DR: It is concluded that the peptides the authors studied containing CRAC sequences are more effective in promoting the formation of cholesterol-rich domains than are shorter peptides of this region of caveolin, which although they contain several aromatic amino acids, they have no CRAC motif.
TL;DR: The classifications of promoter and inhibitor sterols are consistent with previous designations based on fluorescence quenching and detergent resistance and it is found that the vesicle phase behavior is related to the structure of the sterols.
TL;DR: Benecol (margarine; Raisio Group, Raisia, Finland) as mentioned in this paper is a plant sterol and stanol ester that is used to lower LDL cholesterol.
Abstract: Plant stanol esters provide a novel approach to lowering plasma low-density lipoprotein (LDL) cholesterol by dietary means. Their development was preceded by a long period of research into the cholesterol-lowering properties of plant sterols and, recently, plant stanols. Both classes of compound competitively inhibit the absorption of cholesterol and thus lower its level in plasma. Initial impressions were that stanols were more effective and safer than sterols, but the negative outcome of a study led to the recognition that the lipid solubility of free stanols was very limited. This was overcome by esterifying them with fatty acids, with the resultant stanol esters being freely soluble in fat spreads. This led to the launch of Benecol (margarine; Raisio Group, Raisio, Finland) in 1995. The coincident publication of the year-long North Karelia study conclusively demonstrated the long-term LDL-lowering efficacy of plant stanol esters. Variables that might influence the efficacy of stanol esters include dose, frequency of administration, food vehicle in which the stanol ester is incorporated, and background diet. The effective dose is 1 to 3 g/day, expressed as free stanol, which, in placebo-controlled studies, decreased LDL cholesterol by 6% to 15%. This effect is maintained, appears to be similar with once-daily or divided dosage, and is independent of the fat content of the food vehicle. Short-term studies suggest that equivalent amounts of plant sterol and stanol esters are similarly effective in lowering LDL, the main difference being that plasma plant sterol levels increase on plant sterols and decrease on plant stanols. The clinical significance of these changes remains to be determined.
TL;DR: It is proposed that dnpc1a mutants have sterols trapped in aberrant organelles, leading to a shortage of sterol in the endoplasmic reticulum and/or mitochondria of ring gland cells, and, consequently, inadequate ecdysone synthesis.
Abstract: Niemann-Pick type C (NPC) disease is a fatal autosomal-recessive neurodegenerative disorder characterized by the inappropriate accumulation of unesterified cholesterol in aberrant organelles. The disease is due to mutations in either of two genes, NPC1, which encodes a transmembrane protein related to the Hedgehog receptor Patched, and NPC2, which encodes a secreted cholesterol-binding protein. Npc1 mutant mice can be partially rescued by treatment with specific steroids. We have created a Drosophila NPC model by mutating dnpc1a, one of two Drosophila genes related to mammalian NPC1. Cells throughout the bodies of dnpc1a mutants accumulated sterol in a punctate pattern, as in individuals with NPC1 mutations. The mutants developed only to the first larval stage and were unable to molt. Molting after the normal first instar period was restored to various degrees by feeding the mutants the steroid molting hormone 20-hydroxyecdysone, or the precursors of ecdysone biosynthesis, cholesterol and 7-dehydrocholesterol. dnpc1a is normally highly expressed in the ecdysone-producing ring gland. Ring gland-specific expression of dnpc1a in otherwise mutant flies allowed development to adulthood, suggesting that the lack of ecdysone in the mutants is the cause of death. We propose that dnpc1a mutants have sterols trapped in aberrant organelles, leading to a shortage of sterol in the endoplasmic reticulum and/or mitochondria of ring gland cells, and, consequently, inadequate ecdysone synthesis.
TL;DR: Investigating the biochemical modifications occurring in HePC-resistant (HePC-R) Leishmania donovani promastigotes found that the content of unsaturated phospholipid alkyl chains was lower in He PC-R parasite plasma membranes than in those of the wild type, suggesting a lower fluidity of HePC -R parasite membranes.
Abstract: Miltefosine (hexadecylphosphocholine [HePC]) is the first orally active drug approved for the treatment of visceral leishmaniasis. In order to investigate the biochemical modifications occurring in HePC-resistant (HePC-R) Leishmania donovani promastigotes, taking into account the lipid nature of HePC, we investigated their fatty acid and sterol metabolisms. We found that the content of unsaturated phospholipid alkyl chains was lower in HePC-R parasite plasma membranes than in those of the wild type, suggesting a lower fluidity of HePC-R parasite membranes. We also demonstrated that HePC insertion within an external monolayer was more difficult when the proportion of unsaturated phospholipids decreased, rendering the HePC interaction with the external monolayer of HePC-R parasites more difficult. Furthermore, HePC-R parasite membranes displayed a higher content of short alkyl chain fatty acids, suggesting a partial inactivation of the fatty acid elongation enzyme system in HePC-R parasites. Sterol biosynthesis was found to be modified in HePC-R parasites, since the 24-alkylated sterol content was halved in HePC-R parasites; however, this modification was not related to HePC sensitivity. In conclusion, HePC resistance affects three lipid biochemical pathways: fatty acid elongation, the desaturase system responsible for fatty acid alkyl chain unsaturation, and the C-24-alkylation of sterols.
TL;DR: In this article, the authors examined the phytosterol content and composition of various cereal by-products and extracted total lipids in rice bran, wheat bran and wheat germ, wheat germ mixture, durum wheat, oat hull, and corn fine fiber.
Abstract: Phytosterols are hypocholesterolemic. Like corn fiber oil, the lipid extracts of certain cereal by-products may be rich sources of these health-promoting compounds. The objective of this research was to examine the phytosterol content and composition of various cereal by-products. Total lipids in rice bran, wheat bran, wheat germ, durum wheat (bran and germ mixture), oat bran, oat hull, and corn fine fiber were extracted, and the sterol profiles of the extracted lipids were analyzed by GC. Rice bran contained the most lipids (22.2%), followed by wheat germ, durum wheat, oat bran, wheat bran, and oat hull; corn fine fiber contained the least amount of lipids (1.7%). Sitosterol, campesterol, and stigmasterol were the major phytosterols in these lipid extracts, whereas brassicasterol was detected only in wheat samples. Rice bran oil contained considerable amounts of cycloartenol and 24-methylenecycloartanol, which were unique to these samples. Total sterol concentrations in extracted lipids were similar for rice bran, wheat bran, wheat germ, and durum wheat (21.3–15.1 mg/g), but they were very low in oat bran lipids and oat hull lipids (3.4 and 8.2 mg/g, respectively). Corn fine fiber lipids contained the highest amount of sterols (48.3 mg/g). Rice bran appears to be the best source of phytosterols, with the highest oil content and high concentration of sterols.
TL;DR: The present study provides strong evidence that above all satiation and satiety effects underlie the cholesterol-lowering properties of insoluble dietary fibres with moderate or low bile acid-binding capabilities.
Abstract: Fibres with a range of abilities to perturb cholesterol homeostasis were used to investigate how the serum cholesterol-lowering effects of insoluble dietary fibres are related to parameters of intestinal cholesterol absorption and hepatic cholesterol homeostasis in mice. Cholestyramine, chitosan and cellulose were used as examples of fibres with high, intermediate and low bile acid-binding capacities, respectively. The serum cholesterol levels in a control group of mice fed a high fat/high cholesterol (HFHC) diet for 3 weeks increased about 2-fold to 4.3 mm and inclusion of any of these fibres at 7.5 % of the diet prevented this increase from occurring. In addition, the amount of cholesterol accumulated in hepatic stores due to the HFHC diet was reduced by treatment with these fibres. The three kinds of fibres showed similar hypocholesterolaemic activity; however, cholesterol depletion of liver tissue was greatest with cholestyramine. The mechanisms underlying the cholesterol-lowering effect of cholestyramine were (1) decreased cholesterol (food) intake, (2) decreased cholesterol absorption efficiency, and (3) increased faecal bile acid and cholesterol excretion. The latter effects can be attributed to the high bile acid-binding capacity of cholestyramine. In contrast, incorporation of chitosan or cellulose in the diet reduced cholesterol (food) intake, but did not affect either intestinal cholesterol absorption or faecal sterol output. The present study provides strong evidence that above all satiation and satiety effects underlie the cholesterol-lowering properties of insoluble dietary fibres with moderate or low bile acid-binding capabilities.
TL;DR: Findings strongly support the concept that cholesterol absorption is a multistep process, which is regulated by multiple genes at the enterocyte level, and combined therapy using a novel, specific, and potent cholesterol absorption (NPC1L1) inhibitor (ezetimibe) and HMG-CoA reductase inhibitors (statins) offers an efficacious new approach to the prevention and treatment of hypercholesterolemia.
TL;DR: St sterols fulfill two roles in mammalian cells: (i) a bulk membrane requirement in which phytosterols can substitute for cholesterol and (ii) other processes that specifically require small amounts of cholesterol but are not enantioselective.
Abstract: The structural features of sterols required to support mammalian cell growth have not been fully defined. Here, we use mutant CHO cells that synthesize only small amounts of cholesterol to test the capacity of various sterols to support growth. Sterols with minor modifications of the side chain (e.g., campesterol, β-sitosterol, and desmosterol) supported long-term growth of mutant cells, but sterols with more complex modifications of the side chain, the sterol nucleus, or the 3-hydroxy group did not. After 60 days in culture, the exogenous sterol comprised >90% of cellular sterols. Inactivation of residual endogenous synthesis with the squalene epoxidase inhibitor NB-598 prevented growth in β-sitosterol and greatly reduced growth in campesterol. Growth of cells cultured in β-sitosterol and NB-598 was restored by adding small amounts of cholesterol to the medium. Surprisingly, enantiomeric cholesterol also supported cell growth, even in the presence of NB-598. Thus, sterols fulfill two roles in mammalian cells: (i) a bulk membrane requirement in which phytosterols can substitute for cholesterol and (ii) other processes that specifically require small amounts of cholesterol but are not enantioselective.
TL;DR: Several microorganisms and bacterial products target lipid rafts, membrane microdomains of eukaryotic cells enriched in cholesterol, which is a major sterol of mammalian cells.
Abstract: Cholesterol, which is required for viability and cell proliferation, is a major sterol of mammalian cells. More than 90% of cellular cholesterol is located at the plasma membrane. Several microorganisms and bacterial products target lipid rafts, membrane microdomains of eukaryotic cells enriched in
TL;DR: A strong impact of dietary sterols on life history traits and therefore, population dynamics of the keystone species Daphnia is suggested.
Abstract: The absence of essential biochemical nutrients, such as polyunsaturated fatty acids or sterols, has been considered as a mechanism determining trophic interactions between the herbivore Daphnia and its phytoplankton food source. Here, we experimentally quantify the sensitivity of two Daphnia species to decreasing amounts of dietary sterols by measuring variations in life history traits. The two species Daphnia magna and D. galeata were fed different mixtures of the sterol-containing green alga Scenedesmus obliquus and the sterol-free cyanobacterium Synechococcus elongatus; a higher proportion of Synechococcus in the food is equivalent to a decrease in dietary sterols. To address the significance of sterol limitation, the Daphnia species were also fed Synechococcus supplemented with cholesterol. In both species, somatic and population growth rates, maternal dry mass, the number of viable offspring, and the probability of survival were significantly reduced with the lower availability of sterols. A high correlation between the sterol content of the mixed diet and the somatic and population growth rates was found, and growth on cholesterol-supplemented Synechococcus fitted well into this correlation. Somatic growth of first-clutch neonates grown on 100% Synechococcus exhibited a pattern similar to that of somatic growth of their mothers grown on the different food regimes, which demonstrated the significance of maternal effects for sterol-limited population growth. Daphnia galeata had a twofold higher incipient limiting sterol level than D. magna, which indicated interspecific differences in sterol requirements between the two Daphnia species. The results suggest a strong impact of dietary sterols on life history traits and therefore, population dynamics of the keystone species Daphnia.
TL;DR: Plant sterols in the form of their esters when provided in lowfat milk and yoghurt are effective in lowering total and LDL–cholesterol.
Abstract: Background
The cholesterol–lowering efficacy of plant sterol esters (PSteE) or stanol esters (PStaE) in regular– and low–fat spreads has been consistently demonstrated, while their effectiveness in a low–fat, aqueous food carrier such as milk and yoghurt is less well established.
TL;DR: 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.
01 Jan 2005
TL;DR: In this paper, the authors provide a brief review of cholesterol-dependent cytolytic activity of cholesterol dependent cytolysins, commonly known as thiol-activated toxins.
Abstract: Cholesterol, which is required for viability and cell proliferation, is a major sterol of mammalian cells. More than 90% of cellular cholesterol is located at the plasma membrane. Several microorganisms and bacterial products target lipid rafts, membrane microdomains of eukaryotic cells enriched in cholesterol, sphingolipids, and certain proteins. Cholesterol confined in lipid rafts is a crucial component required by microorganisms, directly or indirectly, to enter or exit the intracellular compartment. It is also required for cytolytic activity of cholesterol-dependent cytolysins formerly known as thiol-activated toxins. The object of this review is to provide a brief
TL;DR: The transcriptionalactivation domains of Upc2p and Ecm22p are identified and it is found that UPC2-1, a mutation that allows cells to take up sterols aerobically, increased the potency of the activation domain and the equivalent mutation in ECM22 also greatly enhanced transcriptional activation.
Abstract: Genes encoding biosynthetic enzymes that make ergosterol, the major fungal membrane sterol, are regulated, in part, at the transcriptional level. Two transcription factors, Upc2p and Ecm22p, bind to the promoters of most ergosterol biosynthetic (ERG) genes, including ERG2 and ERG3, and activate these genes upon sterol depletion. We have identified the transcriptional activation domains of Upc2p and Ecm22p and found that UPC2-1, a mutation that allows cells to take up sterols aerobically, increased the potency of the activation domain. The equivalent mutation in ECM22 also greatly enhanced transcriptional activation. The C-terminal regions of Upc2p and Ecm22p, which contained activation domains, also conferred regulation in response to sterol levels. Hence, the activation and regulatory domains of these proteins overlapped. However, the two proteins differed markedly in how they respond to an increased need for sterols. Upon inducing conditions, Upc2p levels increased, and chromatin immunoprecipitation experiments revealed more Upc2p at promoters even when the activation/regulatory domains were tethered to a different DNA-binding domain. However, induction resulted in decreased Ecm22p levels and a corresponding decrease in the amount of Ecm22p bound to promoters. Thus, these two activators differ in their contributions to the regulation of their targets.