Cloning and Characterization of ERG25, the Saccharomyces Cerevisiae Gene Encoding C-4 Sterol Methyl Oxidase (Fungi/Sterol Biosynthesis) M
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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 186-190, January 1996 Genetics Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase (fungi/sterol biosynthesis) M. BARD*t, D. A. BRUNER*, C. A. PIERSON*, N. D. LEES*, B. BIERMANN*, L. FRYEt, C. KOEGEL§, AND R. BARBUCH§ *Department of Biology, Indiana University-Purdue University at Indianapolis, Indianapolis, IN 46202; tDepartment of Chemistry, Rensselaer Polytechnic Institute, Troy, NY 12180; and §Marion Merrell Dow Pharmaceutical Inc., Cincinnati, OH 45215 Communicated by David B. Sprinson, St. Luke's-Roosevelt Hospital Center, New York, NY, July 27, 1995 (received for review June 9, 1995) ABSTRACT We have cloned the Saccharomyces cerevisiae LANOSTEROL C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to ERG1 1 remove the two C-4 methyl groups leading to cholesterol * (animal), ergosterol (fungal), and stigmasterol (plant) bio- 4,4-DIMETHYLCHOLESTA-8,14,24-TRIENOL synthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzy- ; ERG24 mosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 4,4-DIMETHYLZYMOSTEROL 309-amino acid polypeptide (calculated molecular mass, 36.48 ; ERG25, ERG(?) kDa). The amino acid sequence shows a C-terminal endoplas- mic reticulum retrieval signal KKXX and three histidine-rich ZYMOSTEROL clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The ERG6, ERG2, ERG3 sterol profile of an ERG25 disruptant was consistent with the * ERG5, ERG4 erg25 allele obtained by mutagenesis. ERGOSTEROL In the synthesis of sterols, required components of eukaryotic membranes, an initial sterol (lanosterol in animals and fungi FIG. 1. Steps in ergosterol biosynthesis and encoding genes. and cycloartenol in plants) undergoes three demethylations prior to formation of the end product sterol. The first de- genes encoding the enzymes used in C-4 demethylation. In one methylation occurs directly with lanosterol or cycloartenol and report, a Chinese hamster ovary cell line auxotrophic for results in removal of the C-14 methyl group. The fungal cholesterol was observed to accumulate sterol with a C-4 demethylation is performed by the product of the ERG11 gene carboxylic acid group, indicating a deficiency in the decarbox- (Fig. 1). The remaining demethylations occur with the sequen- ylase (7). In addition, the fungal pathogen Cryptococcus neo- tial removal of the two C-4 methyl groups and result in the formans has been shown to accumulate 3-keto sterol after formation of zymosterol in animals and fungi (Fig. 1). exposure to the antifungal itraconazole, thus providing a The steps involved in removal of the C-4 methyl groups are potential screen for isolating strains deficient in 3-keto reduc- not well-defined in yeast but have been described in some tase activity (3, 8). This report describes the isolation of a yeast detail in animals by Gaylor's group (1-3). As depicted in Fig. mutant (erg25), which is auxotrophic for sterol and accumu- 2, the demethylation is specific for the C-4a methyl group and lates 4,4-dimethylzymosterol, indicating a defect in the C-4 is initiated by the C-4 methyl oxidase, which converts the sterol methyl oxidase. Complementation with a yeast genomic methyl group to the alcohol, the aldehyde, and finally to the library has permitted the isolation, sequencing, and disruption carboxylic acid (Fig. 2, structures a-d). This sequential oxida- of the yeast ERG25 gene.l tion was shown to require NADH and oxygen and cytochrome b5 as an electron carrier between the NADH and the oxidase. However, Maitra et al. (4) were unable to show, using 3H MATERIALS AND METHODS release from C-30 4,4-[3H]dimethylzymosterol, that cyto- Strains, Media, Transformation, and Mutagenesis. The chrome b5 was involved in C-4 methyl oxidation. In the next following yeast strains were used: SGY688 (MATa erglA.:URA3 reaction, the carboxyl group is removed by a second enzyme, ura3-52 hem3-11 trpl ade2-11 leu2 lys2), WAl (MATa ura3-52 the C-4 decarboxylase, resulting in formation of a keto group leu2-3,112 adeS his7-2), WA6 (same as WAl but MATa), and at the C-3 position (structure e). A third enzyme, 3-keto upc2 (9). CP2 (MA Ta ergl.:URA3 trp] his3 upc2 ade2-11) was reductase, then reduces the keto group to the required alcohol a segregant from a cross between SGY688 and WA6 followed (structure f). This process is repeated with the second methyl by a cross to upc2, a mutation that allows for aerobic uptake group after its transposition to the a position (5). of sterol. CP2 was plated onto lanosterol-supplemented me- The yeast Saccharomyces cerevisiae has long served as a dium to select for isolates (CP3) capable of specifically utilizing model system for studies in sterol biosynthesis. To this point, this sterol as a substrate for ergosterol synthesis. CP3 was most genes in yeast ergosterol synthesis have been at least grown at 30°C on yeast complete medium (1% yeast ex- partially characterized by direct cloning of the genes or by tract/2% peptone/2% glucose) supplemented with ergosterol analysis of mutations (6). The exceptions to this are the three Abbreviation: ORF, open reading frame. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in $The sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U31885). 186 Downloaded by guest on September 29, 2021 Genetics: Bard et al. Proc. Natl. Acad. Sci. USA 93 (1996) 187 ERG 25 ERG 25 (a) - (b) 4,4-dimethylzymosterol 4-methylzymosterol-4-carboxylic acid 3-keto-4-methylzymosterol 4a-methylzymosterol FIG. 2. Sterol structures and reactions involved in removal of the C-4a methyl group. or an ergosterol intermediate (0.002%) and Tween 80 (0.5%). cells were plated onto ergosterol medium and replica plated to Minimal medium consisted of 0.67% yeast nitrogen base, 2% lanosterol-containing medium. Putative ergosterol mutants glucose, and amino acid and nitrogenous base supplements as could not grow on lanosterol. a 0.8% complete synthetic mixture (CSM) addition (Bio 101). The genomic library of S. cerevisiae in vector YCp5O was Ergosterol and commercial grade lanosterol were purchased obtained from the laboratory of David Botstein (11). Approx- from Sigma. Pure lanosterol (>95%) was obtained from imately 5000 yeast transformants were selected for uracil Steraloids or as a gift from David Nes (Texas Tech). prototrophy on minimal medium lacking uracil (CSM-ura) CP3 yeast cells were grown overnight in complete medium but containing ergosterol and then screened for ergosterol supplemented with ergosterol and mutagenized with ethyl prototrophy on yeast minimal medium without ergosterol. according to standard procedures (10). The Plasmid DNA was extracted from yeast cells by standard methanesulfonate methods (10) and transformed into Escherichia coli DH5a for kill rate for an average experiment was 90-95%. Mutagenized plasmid amplification, restriction digests, and subcloning of DNA fragments. DH5a was grown in LB medium supple- mented with ampicillin (50 mg/liter) and DNA was isolated by A the alkaline lysis method (12) and purified using Qiagen columns. Plasmid Constructions and Gene The 2.4- and 0 Disruption. (I) 3.5-kb BamHI fragments from pERG25-1 were isolated from low melting point agarose gels and subcloned into the yeast 0 vector to generate plasmids pIU704 and pIU705, respectively. pIU707 contains the 1.35-kb Dra I fragment from pIU705 filled in and subcloned into the Sma I site of pRS316 (13). pIU709 contains a 0.4-kb Xba I deletion of pIU705. To disrupt the opening reading frame (ORF) of ERG25, a 1.2-kb Xba I fragment containing the URA3 gene was ligated into the XbaI site of pIU709 to generate pIU802. The 2.2-kb Dra I fragment was excised from a low melting point agarose gel and used to transform the wild-type diploid WA1/6 (14). Segregants were obtained by tetrad analysis. 18 20 22 DNA Sequencing. The 3.5-kb BamHI fragment containing the entire ERG25 gene was cloned into the Bluescript vector Retention Time pBS (KS+) in both orientations. The nucleotide sequences of FIG. 3. GC analyses of sterols accumulating in erg25 grown in the the insert of both strands were determined by the chain- presence of ergosterol. Peak A, ergosterol; peak B, lanosterol; peak C, termination method using Sequenase version 2.0 and deoxy- dimethylzymosterol . adenosine 5'-[a-[35S]thio]triphosphate. Initially, DNA was se- Downloaded by guest on September 29, 2021 188 Genetics: Bard et al. Proc. Natl. Acad. Sci. USA 93 (1996) 10(1, 1 69 Sterol Analyses. Sterols were isolated as nonsaponifiables as described (15). GC analyses of nonsaponifiables were per- formed on a HP5890 series II GC equipped with the HP chemstation software package. The capillary column (HP-5) was 15 m x 0.25 mm x 0.25 jam film thickness and was HO programmed from 195°C to 300°C (3 min at 195°C and then an il increase at 5.5°C/min until the final temperature of 300°C was reached and held for 4 min). The linear velocity was 30 cm/sec using nitrogen as the carrier gas, and all injections were run in () the splitless mode. GC/MS analyses were done with a Varian 3400 gas chromatograph interfaced to a Finnigan MAT TSQ II 700 mass spectrometer. The GC separations were done on a fused silica column, DB-5 15 m x 0.32 mm x 0.25 ,um film 412 thickness, programmed from 50°C to 250°C at 20°C/min after ,' 1 a 1-min hold at 50°C.