A novel gene conserved from yeast to humans is involved in sterol biosynthesis.

The ERG28 gene was originally identified by microarray expression profiling as possibly involved in the Saccharomyces cerevisiae sterol pathway. Microarray analyses suggested that the transcription pattern of ERG28 closely followed that of genes involved in sterol synthesis. ERG28 was also found in Schizosaccharomyces pombe and Arabidopsis as well as humans, and in the latter was shown to be highly expressed in adult testis tissue. All four proteins contain potential transmembrane domain(s). Gas chromatography-mass spectrometry analysis of an ERG28-deleted S. cerevisiae strain (which is slow growing but not auxotrophic for ergosterol) indicates a lesion in sterol C-4 demethylation. Sterol profiles indicate accumulation of 3-keto and carboxylic acid sterol intermediates, which are involved in removing the two C-4 methyl groups from the sterol A ring. Similar intermediates have previously been demonstrated to accumulate in erg26 (sterol dehydrogenase/decarboxylase) and erg27 (3-ketoreductase) mutants in yeast. We speculate that the role of the Erg28 protein (Erg28p) may be either to tether Erg26p and Erg27p to the endoplasmic reticulum or to facilitate interaction between these proteins.-Gachotte, D., J. Eckstein, R. Barbuch, T. Hughes, C. Roberts, and M. Bard. A novel gene conserved from yeast to humans is involved in sterol biosynthesis. J. Lipid Res. 2001. 42: 150;-154.

Functional discovery via a compendium of expression profiles.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation.

The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned. This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position. We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol. A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates. Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methyl-fecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fifth intermediate was isolated and identified by (1)H NMR as a 4-methyl-24, 25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.

Delta7-sterol-C5-desaturase: molecular characterization and functional expression of wild-type and mutant alleles.

An Arabidopsis thaliana recessive monogenic mutant (ste1-1) presenting a deficiency of the delta7-sterol-C5(6)-desaturase step in the sterol pathway has been reported previously [12]. To further characterize ste1-1, Arabidopsis, Nicotiana tabacum and Homo sapiens cDNAs encoding delta7-sterol-C5(6)-desaturases were isolated and identified on the basis of their ability to restore ergosterol synthesis in erg3, a yeast null mutant whose gene encoding the delta7-sterol-C5(6)-desaturase was disrupted. Overexpression of the Arabidopsis cDNA driven by a 35S promoter in transgenic ste1-1 plants led to full complementation of the mutant. This result demonstrates that STE1 was the impaired component in the desaturation system. Four independent reverse transcriptions of ste1-1 RNA followed by polymerase chain reactions (RT-PCRs), yielded a single product. Alignment of the wild-type ORF with the RT-PCR derived ste1-1 ORF revealed a single amino acid substitution: Thr-114 in the wild-type is changed to Ile in ste1-1. Expression in erg3 resulted in a 6-fold lowered efficiency of the ste1-1 ORF in complementing the yeast biosynthetic pathway when compared to the wild-type ORF. The presence of this mutation in the mutant ste1-1 genomic sequence (and no additional modification between ste1-1 and wild-type genes) demonstrates that the change of the Thr-114 to Ile is necessary and sufficient to create the leaky allele ste1-1. The occurrence of a hydroxylated amino acid (Thr or Ser) at the position corresponding to Thr-114 in the five delta7-sterol-C5(6)-desaturases identified so far suggests that this amino acid is important for normal enzymatic function.

Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis.

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Delta strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4beta, 14alpha-dimethyl-4alpha-carboxy-cholesta-8,24-dien-3be ta-ol and 4beta-methyl-4alpha-carboxy-cholesta-8,24-dien-3beta-o l, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.

A yeast sterol auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme.

Genetic disruption of the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxotrophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is erg11, which is blocked in 14alpha-demethylation of lanosterol and is itself an auxotroph. The second suppressor mutation required is either slu1 or slu2 (suppressor of lanosterol utilization). These mutations are leaky versions of HEM2 and HEM4, respectively; addition of exogenous hemin reverses the suppressing effects of slu1 and slu2. Suppression of erg25 by erg11 slu1 (or erg11 slu2) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates. This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth. In the triple mutants, all but 1 (ERG6) of the 13 subsequent reactions of the ergosterol pathway are inactive. Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the ERG11 gene product, the 14alpha-demethylase. In this investigation, we demonstrate that treatment of the sterol auxotrophs erg25 slu1 or erg25 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system we have described.

Isolation and characterization of an Arabidopsis thaliana cDNA encoding a delta 7-sterol-C-5-desaturase by functional complementation of a defective yeast mutant.

A yeast null mutant (erg 3) defective in ERG 3, the gene encoding the C-5 sterol desaturase required for ergosterol synthesis was transformed with an Arabidopsis thaliana cDNA library inserted in a yeast vector. Transformants (4 x 10(5)) were screened for cycloheximide (CH) resistance and 400 possible clones were analyzed to determine their sterol profile. Low levels of ergosterol in addition to delta 7- and delta 8-sterols normally present in erg3 were isolated in three yeast transformants. Characterization of one transformant indicated a cDNA of 1141 bp. Transformation of an erg 3 strain with this plasmid led to CH resistance, nystatin sensitivity and an ergosterol profile. After subcloning in a pBluescript vector and subsequent sequencing, an ORF of 843 bp encoding a possible 281 amino acid polypeptide was deduced. Three histidine-rich motifs (HX3H, HX2HH and HX2HH) were found in the A. thaliana ORF which are also present in the yeast ERG 3 gene. These histidine-rich motifs are also characteristic of many membrane-bound fatty acid desaturases from higher plants. These data strongly suggest that the A. thaliana cDNA encodes a delta 7-sterol-C-5-desaturase.

Transformation of Saccharomyces cerevisiae with a cDNA encoding a sterol C-methyltransferase from Arabidopsis thaliana results in the synthesis of 24-ethyl sterols.

Using an EST-cDNA probe, a full-length cDNA (411) sequence of 1411 bp was isolated from A. thaliana. This sequence contained features typical of methyltransferases in general and in particular showed 38% identity with ERG6, a S. cerevisiae gene which encodes the zymosterol-C-24-methyltransferase. A yeast vector containing this ORF (4118-pYeDP60) was used to transform a wild type S. cerevisiae which accumulates predominantly ergosterol, a 24-methyl sterol as well as a mutant erg6 null mutant accumulating principally zymosterol, a sterol non-alkylated at C-24. In both cases, several 24-ethyl- and 24-ethylidene sterols were synthetized indicating that the 4118 cDNA encodes a plant sterol C-methyltransferase able to perform two sequential methylations of the sterol side chain.

An Arabidopsis mutant deficient in sterol biosynthesis: heterologous complementation by ERG 3 encoding a delta 7-sterol-C-5-desaturase from yeast.

The mutant STE 1 was isolated by screening an ethylmethane sulfonate (EMS)-mutagenized population of Arabidopsis thaliana which consisted of 22,000 M2 plants divided into 1100 pools of 20 plants by gas chromatography of sterols extracted from small leaf samples. STE 1 was characterized by the accumulation of three delta 7-sterols concomitantly with the decrease of the three corresponding delta 5-sterols which are the end products of the sterol pathway in wild-type leaves. The structure of these delta 7-sterols was determined after two steps of purification on HPLC, by gas chromatography coupled with mass spectrometry (GC-MS) and proton nuclear magnetic resonance spectrometry (1H-NMR). The accumulation of delta 7-sterols suggested that the mutant is deficient in the activity of the delta 7-sterol-C-5-desaturase. Genetic analysis showed that the accumulation of delta 7-sterols was due to a single recessive nuclear mutation. The mutant line STE 1 was backcrossed four times to the wild-type. The resulting STE 1 plants had wild-type morphology and set seeds normally, suggesting that the delta 7-sterols in STE 1 are good surrogates of physiologically active delta 5-sterols to sustain normal development. STE 1 roots were transformed with the Saccharomyces cerevisiae ERG 3 gene encoding the delta 7-sterol-C-5-desaturase under the control of the CaMV 35S promoter. Seven transgenic STE 1 root-derived calli showed an increase in delta 5-sterols and a concomitant decrease in delta 7-sterols in comparison with STE 1 untransformed root-derived calli. Northern blot analysis using the ERG 3 probe showed a strong expression of ERG 3 in three of the seven transgenic calli. These results suggest that the accumulation of delta 7-sterols in the STE 1 mutant is due to a deficiency of the delta 7-sterol-C-5-desaturation step in the plant sterol biosynthesis pathway.