The effects of the hypocholesteremic compound 3β‐(β‐dimethylaminoethoxy)‐androst‐5‐en‐17‐one on the sterol and steryl ester composition of<i>Saccharomyces cerevisiae</i>

Oxidosqualene cyclase of the yeast encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study (Athenstaedt, K., Zweytick, D., Jandrositz, A, Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441-6448), Erg7p was identified as a component of yeast lipid particles. Here, we present evidence that Erg7p is almost exclusively associated with this compartment as shown by analysis of enzymatic activity, Western blot analysis, and in vivo localization of Erg7p-GFP. Occurrence of oxidosqualene cyclase in other organelles including the endoplasmic reticulum is negligible. In an erg7 deletion strain or in wild-type cells treated with an inhibitor of oxidosqualene cyclase, the substrate of Erg7p, oxidosqualene, accumulated mostly in lipid particles. Storage in lipid particles of this intermediate produced in excess may provide a possibility to exclude this membrane-perturbing component from other organelles. Thus, our data provide evidence that lipid particles are not only a depot for neutral lipids, but also participate in coordinate sterol metabolism and trafficking and serve as a storage site for compounds that may negatively affect membrane integrity. Oxidosqualene cyclases (OSCs)1 play a central role in sterol biosynthesis because they catalyze a key reaction of the sterol biosynthetic pathway, namely formation of a cyclic component from the acyclic precursors oxidosqualene and dioxidosqualene ( Fig. 1). OSCs may be regarded as phylogenetic markers insofar as they form a variety of cyclic triterpenoids in plants, whereas their only cyclization product in nonphotosynthetic eukaryotes such as animals, yeast, and other fungi is lanosterol (1). During the last 20 years studies on OSCs focused on several biochemical, molecular biological, and cell biological aspects.Hundreds of inhibitors were synthesized and tested for their effect on this enzyme (2-6), OSCs from different sources were characterized and purified (7-10), and genes encoding these proteins in various species were cloned and sequenced (11)(12)(13)(14)(15). During these studies, however, the important question as to the subcellular localization of this enzyme was surprisingly ignored. This aspect is essential because localization of enzymes involved in the sterol biosynthetic pathway may contribute greatly to the understanding of coordination and regulation of this pathway and of sterol traffic among organelles (16 -19).Subcellular localization of yeast OSC, which is encoded by the ERG7 gene, stimulated our interest not only because Erg7p is one of the most important enzymes of sterol biosynthesis that has not been localized so far, but also because targeting of the protein to its proper cellular environment may help to explain its mode of action and to design new inhibitors of OSC. As an example, we reported recently that some OSC inhibitors that undergo metabolic transformations in the yeast exhibit different effects because of different subcellular distribution of target protei...

Section: Methodsmentioning

confidence: 99%

Yeast Oxidosqualene Cyclase (Erg7p) Is a Major Component of Lipid Particles

Milla¹,

Athenstaedt²,

Viola³

et al. 2002

“…14 C]mevalonate as described (44), in the presence of 0.1 mM of the oxidosqualene cyclase (OSC) inhibitor U14266A (U14, 3␤-(2-dimethylaminoethoxy)-androst-5-on-17-one) (45). After 3 h of incubation, the reaction was stopped by adding 1 volume of 15% KOH in methanol, and lipids were saponified at 80°C for 30 min.…”

Section: Metabolic Radiolabeling and Analysis Of Sterols And Neutral mentioning

confidence: 99%

Characterizing Sterol Defect Suppressors Uncovers a Novel Transcriptional Signaling Pathway Regulating Zymosterol Biosynthesis

Germann

Gallo

Donahue

et al. 2005

erg26-1ts cells harbor defects in the 4␣-carboxysterol-C3 dehydrogenase activity necessary for conversion of 4,4-dimethylzymosterol to zymosterol. Mutant cells accumulate toxic 4-carboxysterols and are inviable at high temperature. A genetic screen aimed at cloning recessive mutations remediating the temperature sensitive growth defect has resulted in the isolation of four complementation groups, ets1-4 (erg26-1 ts temperature sensitive suppressor). We describe the characterization of ets1-1 and ets2-1. Gas chromatography/mass spectrometry analyses demonstrate that erg26-1 ts ets1-1 and erg26-1 ts ets2-1 cells do not accumulate 4-carboxysterols, rather these cells have increased levels of squalene and squalene epoxide, respectively. ets1-1 and ets2-1 cells accumulate these same sterol intermediates. Chromosomal integration of ERG1 and ERG7 at their loci in erg26-1 ts ets1-1 and erg26-1 ts ets2-1 mutants, respectively, results in the loss of accumulation of squalene and squalene epoxide, re-accumulation of 4-carboxysterols and cell inviability at high temperature. Enzymatic assays demonstrate that mutants harboring the ets1-1 allele have decreased squalene epoxidase activity, while those containing the ets2-1 allele show weakened oxidosqualene cyclase activity. Thus, ETS1 and ETS2 are allelic to ERG1 and ERG7, respectively. We have mapped mutations within the erg1-1/ ets1-1 (G247D) and erg7-1/ets2-1 (D530N, V615E) alleles that suppress the inviability of erg26-1 ts at high temperature, and cause accumulation of sterol intermediates and decreased enzymatic activities. Finally using erg1-1 and erg7-1 mutant strains, we demonstrate that the expression of the ERG25/26/27 genes required for zymosterol biosynthesis are coordinately transcriptionally regulated, along with ERG1 and ERG7, in response to blocks in sterol biosynthesis. Transcriptional regulation requires the transcription factors, Upc2p and Ecm22p.

Section: Partial Inhibition Of Oxidosqualene-lanosterol Cyclase Enhanmentioning

confidence: 99%

“…inhibition that does not compromise the growth of the cells and that still allows production of ergosterol, results in an increase in the cellular levels of 2,(3S)-oxidosqualene, 2,(3S),(22S),23-dioxidosqualene, and an unidentified molecule with greater polarity than ergosterol, likely an oxysterol molecule, with a concomitant decrease in ergosterol and ergosterol esters (33,36,49). Enzymatic conversion of 2,(3S),(22S),23-dioxidosqualene to 24,25-oxidolanosterol by oxidosqualene-lanosterol cyclase could be observed by incubation of the cells under anaerobic conditions (49). Otherwise, incubation of cells under aerobic conditions results in the enzymatic conversion of 2,(3S),(22S),23-dioxidosqualene to an unidentified, putative oxysterol molecule (49), consistent with the requirement of oxygen for enzymatic actions directly downstream of oxidosqualene-lanosterol cyclase in the sterol/oxysterol biosynthetic pathways.…”

Section: Partial Inhibition Of Oxidosqualene-lanosterol Cyclase Enhanmentioning

confidence: 99%

“…Enzymatic conversion of 2,(3S),(22S),23-dioxidosqualene to 24,25-oxidolanosterol by oxidosqualene-lanosterol cyclase could be observed by incubation of the cells under anaerobic conditions (49). Otherwise, incubation of cells under aerobic conditions results in the enzymatic conversion of 2,(3S),(22S),23-dioxidosqualene to an unidentified, putative oxysterol molecule (49), consistent with the requirement of oxygen for enzymatic actions directly downstream of oxidosqualene-lanosterol cyclase in the sterol/oxysterol biosynthetic pathways. These previous observations suggest that the down-regulation or inhibition of oxidosqualenelanosterol cyclase we employed above to examine Hmg2p degradation likely resulted in the production of 2,(3S),(22S),23- Because downstream products of the alternate pathway are produced in yeast (33,36,49), and partial inhibition or downregulation of oxidosqualene-lanosterol cyclase resulted in enhanced Hmg2p degradation (see above), we tested if complete inhibition of oxidosqualene-lanosterol cyclase had an additional enhancing effect on Hmg2p degradation, consistent with the substrate of oxidosqualene-lanosterol cyclase acting as a positive signal for HMGR degradation, or if complete inhibition was without effect on Hmg2p degradation, consistent with an oxysterol-derived molecule acting as a positive signal for HMGR degradation.…”

Section: Partial Inhibition Of Oxidosqualene-lanosterol Cyclase Enhanmentioning

confidence: 99%

See 1 more Smart Citation

An Oxysterol-derived Positive Signal for 3-Hydroxy- 3-methylglutaryl-CoA Reductase Degradation in Yeast

Gardner

Shan²,

Matsuda

et al. 2001

Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In mammals, both a non-sterol isoprenoid signal derived from farnesyl diphosphate (FPP) and a sterol-derived signal appear to act together to positively regulate the rate of HMGR degradation. Although the nature and number of sterol-derived signals are not clear, there is growing evidence that oxysterols can serve in this capacity. In yeast, a similar non-sterol isoprenoid signal generated from FPP acts to positively regulate HMGR degradation, but the existence of any sterol-derived signal has thus far not been revealed. We now demonstrate, through the use of genetic and pharmacological manipulation of oxidosqualene-lanosterol cyclase, that an oxysterol-derived signal positively regulated HMGR degradation in yeast. The oxysterol-derived signal acted by specifically modulating HMGR stability, not endoplasmic reticulum-associated degradation in general. Direct biochemical labeling of mevalonate pathway products confirmed that oxysterols were produced endogenously in yeast and that their levels varied appropriately in response to genetic or pharmacological manipulations that altered HMGR stability. Genetic manipulation of oxidosqualene-lanosterol cyclase did result in the buildup of detectable levels of 24,25-oxidolanosterol by gas chromatography, gas chromatography-mass spectroscopy, and NMR analyses, whereas no detectable amounts were observed in wild-type cells or cells with squalene epoxidase down-regulated. In contrast to mammalian cells, the yeast oxysterol-derived signal was not required for HMGR degradation in yeast. Rather, the function of this second signal was to enhance the ability of the FPP-derived signal to promote HMGR degradation. Thus, although differences do exist, both yeast and mammalian cells employ a similar strategy of multiinput regulation of HMGR degradation.