Transcriptional regulation of the squalene synthase gene (ERG9) in the yeast Saccharomyces cerevisiae

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Transcriptional regulation of the squalene synthase gene (ERG9) in the yeast Saccharomyces cerevisiae Matthew A. Kennedy a , Robert Barbuch b , Martin Bard a

a;

*

Department of Biology, Indiana University-Purdue University at Indianapolis, 723 W. Michigan Street, SL324, Indianapolis, IN 46202, USA b Hoechst Marion Roussel Inc., Cincinnati, OH 45215, USA Received 8 February 1999; accepted 11 February 1999

Abstract The ergosterol biosynthetic pathway is a specific branch of the mevalonate pathway. Since the cells requirement for sterols is greater than for isoprenoids, sterol biosynthesis must be regulated independently of isoprenoid biosynthesis. In this study we explored the transcriptional regulation of squalene synthase (ERG9) in Saccharomyces cerevisiae, the first enzyme dedicated to the synthesis of sterols. A mutant search was performed to identify genes that were involved in the regulation of the expression of an ERG9-lacZ promoter fusion. Mutants with phenotypes consistent with known sterol biosynthetic mutations (ERG3, ERG7, ERG24) increased expression of ERG9. In addition, treatment of wild-type cells with the sterol inhibitors zaragozic acid and ketoconazole, which target squalene synthase and the C-14 sterol demethylase respectively, also caused an increase in ERG9 expression. The data also demonstrate that heme mutants increased ERG9 expression while anaerobic conditions decreased expression. Additionally, the heme activator protein transcription factors HAP1 and HAP2/ 3/4, the yeast activator protein transcription factor yAP-1, and the phospholipid transcription factor complex INO2/4 regulate ERG9 expression. ERG9 expression is decreased in hap1, hap2/3/4, and yap-1 mutants while ino2/4 mutants showed an increase in ERG9 expression. This study demonstrates that ERG9 transcription is regulated by several diverse factors, consistent with the idea that as the first step dedicated to the synthesis of sterols, squalene synthase gene expression and ultimately sterol biosynthesis is highly regulated. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Ergosterol; Squalene synthase; Transcription; Regulation ; Promoter; (Saccharomyces cerevisiae)

1. Introduction Sterols are essential cellular molecules which function to regulate membrane £uidity, permeability, the activity of membrane bound enzymes and growth rate. Ergosterol is the predominant fungal sterol

* Corresponding author. Fax: +1 (317) 274-2846; E-mail: [email protected]

and is equivalent to cholesterol in animals and sitosterol in plants as the end product sterol. The yeast biosynthetic pathway leading to the formation of ergosterol is now well de¢ned [1]. However, the regulation of sterol biosynthesis in yeast has not been well characterized. Squalene synthase (ERG9) is a branch point enzyme in the mevalonate pathway (Fig. 1). AcetylCoA is converted to mevalonate and subsequently to farnesyl pyrophosphate (FPP) in which FPP is either utilized in the formation of dolichols, ubiqui-

0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 0 3 5 - 4

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Fig. 1. The ergosterol biosynthetic and esteri¢cation pathway of Saccharomyces cerevisiae. Genes and their corresponding enzymes discussed in the text are indicated, as is the site of action for two ergosterol biosynthetic inhibitors. Arrows without gene designations represent steps not addressed in this study.

none, heme A, the isoprenylation pathway or the sterol biosynthetic pathway. Sterol formation is dependent on the condensation of two molecules of FPP into squalene by squalene synthase (ERG9) and is thus referred to as the ¢rst dedicated step in sterol biosynthesis which has been conserved among all phyla. Previous investigators have provided evidence that squalene synthase activity is regulated in yeast [2,3]. Overexpression of ERG9 on a 2W plasmid yielded a two-fold increase in squalene synthase activity [2]. In another study, squalene synthase activity was increased up to ¢ve-fold in sterol auxotrophs supplemented with limiting amounts of sterol and de-

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creased two-fold under semi-anaerobic conditions [3]. Others found that inhibition of sterol biosynthesis with lovastatin resulted in a ¢ve-fold increase in squalene synthase mRNA levels in Saccharomyces cerevisiae and cultured human ¢broblasts [4]. In Tetrahymena, squalene synthase activity is repressed by the addition of exogenous sterol [5]. In the transfected human hepatoma cell line HepG2 or hamster CHO-K1 cells, human squalene synthase has been shown to be transcriptionally regulated [6]. In the present study, the transcriptional regulation of squalene synthase (ERG9) was examined by expression of the Escherichia coli lacZ gene under the control of the ERG9 promoter in wild-type and mutant strains of S. cerevisiae. In addition, a genetic screen was performed to identify mutants that caused an increase in ERG9 expression. We demonstrate that ERG9 is subject to transcriptional regulation by mutations in the ergosterol biosynthetic pathway that alter the intracellular sterol composition but not by mutants in sterol esteri¢cation. Moreover, interruptions of the sterol pathway leading to decreased squalene synthesis or interconversion of squalene into sterols showed the greatest amount of transcriptional regulation. The data will also demonstrate that ERG9 expression is responsive to intracellular levels of oxygen and heme both directly and in association with the HAP transcription factor complexes. Furthermore, mutants in trans-acting factors involved in stress and phospholipid biosynthesis regulate the expression of ERG9. 2. Materials and methods 2.1. Strains, growth conditions, and transformation methods BWG1-7a (MATa, ade1-100, his4-519, leu2-3,-112, ura3-52) and the HAP mutants LPY22 (hap1v BLEU2), LWG1 (hap2-1), and SHY40 (hap3BHIS4) were provided by L. Guarente [7,8]. Strains disrupted for the C-8 sterol isomerase (erg2BLEU2), the C-5 sterol desaturase (erg3BLEU2), the C-24 sterol reductase (erg4BLEU2), the C-22 sterol desaturase (erg5BLEU2) and the C-24 sterol methyltransferase (erg6BLEU2) have been previously described [9] and are isogenic to BWG1-7a. RZ53-6 (MATK, trp1-289,

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leu2-3,-112, ura3-52, ade1-100), and the hypoxic gene repressor mutant RZ53-6rox1 (rox1vBLEU2) were provided by R. Zitomer [10]. JRY527 (MATa, ura3-52, his3v200, ade2-101, lys2-801, met3 ) and the HMG-CoA reductase mutants JRY1159 (hmg1BLYS2) and JRY1160 (hmg2BHIS3) were provided by J. Rine [11]. Wild-type and ARE (acat-related enzyme) mutants 2D (are1BHIS3), 2C (are2BLEU2), and 2B (are1BHIS3, are2BLEU2) have been previously described [9]. The heme biosynthetic mutant TKY22 (MATa, leu2-3,-112, ura3-52, ade1, trp1BhisG, hem1v) was provided by T. Keng [12]. SEY6210 (MATK, leu2-3,-112, ura3-52, his3v200, trp1v-901, lys2-801, suc2-v9, met3 ) and the YAP mutant SM10 (yap1-v1BHIS3) were provided by W.S. Moye-Rowley [13]. F113 (MATa, ura3-52, canr ) and the amino acid metabolism mutant F212 (gcn4v1) were provided by R. Wek (Indiana School of Medicine, Indianapolis, IN). SH2 (MATa, trp1v63, his3v200, ura3-52, leu2v-1) and the phospholipid mutants SH3 (ino2vBTRP1), SH4 (opi1vBLEU2), and SH7 (ino4vBLEU2) were provided by S. Henry (Carnegie Mellon University, Pittsburgh, PA). H1716 (MATa, gcn4v, his3-609, leu2-3,-112, ura3-52, ino1-13) was provided by R. Rolfes (Massachusetts Institute of Technology, Cambridge, MA), and was used to construct MKY2A (ERG9 promoter-LacZ and ERG9 promoter-HIS3 fusions). Yeast strains were grown at 30³C in YEPD medium containing 2% glucose or complete synthetic medium (SC) medium containing either 2% glucose or 2% galactose with appropriate nutrients omitted as required to maintain plasmid selection [14]. For semi-anaerobic growth conditions, yeast cells were grown in SC media with aeration at 30³C to a density of 2U106 cells/ml (OD660 = 0.2). The cultures were then sealed and nitrogen was bubbled into the culture through a glass pipette inserted through the stopper for 4^6 h at 30³C and 90 rpm to a density of 1.0^1.3U107 cells/ml (OD660 = 0.7^0.8). To test for ergosterol biosynthetic mutants, nystatin (Ny), was added to YEPD after autoclaving and cooling to 55³C to ¢nal concentrations of 2.5, 5, 7.5 and 10 Wg/ml. To select for yeast mutants that were over-expressing histidine, 3-amino-1,2,4-triazole (3AT), was added to appropriate SC media to concentrations of 2, 5, 10 and 20 mM. Ergosterol biosyn-

thetic inhibitors used to examine ERG9 expression were added to the growth media as described by Sprague et al. [15] with minor modi¢cations. Fresh cultures were grown in SC media with 5 WM zaragozic acid A [16] or 1 WM ketoconazole [17] (which reduced the growth rate by 50%), to a density of 5U106 cells/ml (OD660 = 0.4). Zaragozic acid A was then added to a ¢nal concentration of 60 WM and ketoconazole was added to a ¢nal concentration of 10 WM. The cultures were then incubated at 30³C for 15^18 h before harvesting. Yeast strains were transformed using the Lithium acetate method [18]. The E. coli strain DH5K (F3 , P80dlacZvM15 v(lacZYAargF) U169, deoR, recA1, endA1, phoA, ‡ 3 hsdR17(r3 k ,mk ), supE44V , thi-1, gyrA96, relA1) was transformed as previously described by Ausubel [19] and grown in Luria-Bertani (LB) media with 50 Wg/ml ampicillin at 37³C. 2.2. Construction of ERG9 promoter gene fusions and mutagenesis The ERG9 promoter was ampli¢ed by PCR using an ERG9 plasmid obtained from F. Spencer (Johns Hopkins University, Baltimore, MD), as template DNA and the sequence speci¢c primers MB-MK-1 (5P-GGGGGGGATCCATTGTGTGTGTGTGATATGTGACGT-3P) and MB-MK-2 (5P-GGGGGGAAT T C GTACCGTCACAATGTAGGGCTATATAT-3P). MB-MK-1 and MB-MK-2 contained a BamHI and an EcoRI restriction enzyme recognition sequence respectively, which facilitated subcloning of the ampli¢ed DNA into the BamHI and EcoRI sites of the lacZ containing vector, pYLZ-6 [20], to yield pIU850. pYLZ-6 contains the lacZ gene lacking the ¢rst eight coding amino acids adjacent to a multiple cloning site allowing for insertion of in-frame promoter fragments to drive expression of lacZ. Primers were designed to amplify 756 base pairs of the ERG9 promoter. Veri¢cation of the PCR sequence was accomplished by using the Sequenase 2.0 DNA sequencing kit. Sequencing primers were: YLZ6-1 (5P-CAATACGCAAACCGCCTG-3P) and YLZ6-2 (5P-AGGCGATTAAGTTGGGTA-3P) and are homologous to pYLZ-6 vector sequences £anking the ERG9 promoter insert. To construct an integrating ERG9 promoter-lacZ fusion vector, the 1.7 kb ScaI CEN6, ARSH4 frag-

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ment of pIU850 was replaced with the 1.2 kb ScaI fragment of the integrating vector pRS306 [21] to yield pIU850-I. pIU850-I was linearized with either ApaI or StuI in the URA3 gene. Integration was accomplished by transformation and selection for uracil prototrophy. To construct a double ERG9 promoter fusion strain, H1716 (described in Section 2.1) was transformed to uracil prototrophy by integration of pIU850-I at the URA3 locus to yield the strain MKY1A. A second ERG9 promoter fusion was constructed using the HIS3 gene in the following manner. The HIS3 gene was ampli¢ed by PCR using yDp-H [22] as the template DNA and the sequence speci¢c primers HIS3-1 (5P-AGGCAAAGATGGATCCGCAGAAAGCC-3P) and HIS3-2 (5P-GGGCTCCGGGGCAAGAGAAAAAAAAA-3P). HIS3-1 and HIS3-2 contained a BamHI and an EagI restriction enzyme recognition site respectively, which facilitated subcloning of the 800 base pair HIS3 PCR product into the BamHI-EagI site of pRS316 [21] to yield pIU569. The 0.76 kb EcoRI-BamHI ERG9 promoter fragment was subcloned from pIU850 into the EcoRI-BamHI sites of pIU569 to yield pIU890. Ligation of the 3P end of the ERG9 promoter to the 5P end of the HIS3 gene at the BamHI resulted in an inframe fusion. Upon transformation, pIU890 was capable of conferring prototrophy to histidine auxotrophs whereas pIU569, the promoterless HIS3 gene, could not. To make an integrating vector, the ERG9 promoter-HIS3 fusion was subcloned from pIU890 as a 1.6 kb XhoI-EagI fragment into the XhoI-EagI sites of the yeast integrating vector, pRS305 [21] to yield pIU892. pIU892 was linearized within the LEU2 gene with A£II and integrated into the leu2 locus of the yeast strain MKY1A to yield MKY2A, converting the strain to leucine prototrophy. Mutagenesis was performed on MKY2A by UV irradiation by standard methods described by Rose et al. [23] to a survival of 20%. 2.3. Quantitative and qualitative L-galactosidase enzyme assays Quantitative liquid assays were done as described by Rose and Botstein [24]. In vitro speci¢c activity of the L-galactosidase enzyme was measured as a function of nmol of ONPG hydrolyzed/min/mg of pro-

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tein. Protein concentrations were determined using the Bradford assay [25]. Assays were performed on two independent transformants in duplicate over three days and all L-galactosidase values were averaged. A GCN4-promoter-lacZ fusion plasmid [26] was used as a control to verify that the regulatory e¡ect was speci¢c for ERG9. Qualitative ¢lter/colony lift assays were done as described by Vojek [27]. 2.4. Sterol analyses Total sterols were isolated as non-saponi¢ables as described by Molzahn and Woods [28]. Sterols were separated on a Hewlett-Packard 5890 series II gas chromatograph (GC) equipped with the HP chemstation software package. The capillary column (HP-5) was 15 mU0.25 mmU0.25 Wm ¢lm thickness and was programmed from 195 to 300³C (3 min at 195³C and then an increase of 5.5³C/min until the ¢nal temperature of 300³C was reached and held for 4 min). The linear velocity was 30 cm/s using nitrogen as the carrier gas, and all injections were run in the splitless mode. To determine the percentage of total cellular mass represented by sterol, quantitative sterol analysis was performed as described by Woods [29] and sterols were separated by GC analysis as described above. To calculate the percentage of sterol per mg of cell mass, 10 ml of the original culture was harvested by vacuum ¢ltration onto a preweighed 0.45 Wm nitrocellulose ¢lter. The cells were dried in a 75³C oven overnight and weighed. Thin layer chromatography (TLC) was performed as previously described by Kelly et al. [30]. Gas chromatography/Mass spectrometry (GC/MS) analyses of sterols were done with a Varian 3400 gas chromatograph interfaced to a Finnigan MAT TSQ 700 mass spectrometer. The GC separations were done on a fused silica column, DB-5, 15mU0.32 mmU0.25 Wm ¢lm thickness, programmed from 50 to 250³C at 20³C/min after a 1 min hold at 50³C. The oven temperature was held at 250³C for 10 min before programming the temperature to 300³C at 20³C/ min. Helium was the carrier gas with a linear velocity of 50 cm/s in the splitless mode. The mass spectrometer was in the electron impact ionization mode at an electron energy of 70 eV, an ion source temperature of 150³C, and scanning from 40 to 650 atomic mass units at 0.5 s intervals.

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2.5. Materials 3-Amino-1,2,4-triazole (3-AT) was purchased from Fluka (Ronkonkoma, NY) and 5-bromo-4-chloro-3indolyl-L-D-galctopyranoside (X-Gal) was purchased from Diagnostic Chemicals (Oxford, CT). Taq polymerase was purchased from Promega (Madison, WI). The alkali cation yeast transformation kit, the GENECLEAN DNA puri¢cation kit, complete synthetic dropout media and Luria Broth were purchased from BIO 101 (Vista, CA). All other media were purchased from Difco (Detroit, MI). Whatman chromatography paper, nylon and nitrocellulose ¢lters, and all solvents at the GC grade were purchased from Fisher (Pittsburgh, PA). The Bradford protein assay kit was purchased from Bio-Rad Laboratories (Richmond, CA) and the Sequenase 2.0 DNA sequencing kit was purchased from United States Biochemicals (Cleveland, OH). Zaragozic acid A was a gift from M. Kurtz (Merck, Rahway, NJ). All other chemicals were purchased from Sigma (St. Louis, MO). 3. Results and discussion 3.1. Lesions in the ergosterol biosynthetic pathway increase ERG9 expression Squalene synthase (ERG9) is the ¢rst enzyme of the mevalonate pathway dedicated to sterol biosynthesis. We were interested in determining whether a lesion upstream, downstream, or at the ERG9 step would e¡ect the expression of ERG9. To examine this, a panel of mutants and the isogenic wild-type strain were transformed with pIU850 (wild-type ERG9 promoter-lacZ fusion gene) and assayed for L-galactosidase activity under aerobic growth conditions. To assess the e¡ect of a lesion in two genes essential in sterol biosynthesis, the ergosterol biosynthetic inhibitors zaragozic acid and ketoconazole were used to inhibit ergosterol synthesis. As indicated in Fig. 1, the site of action for zaragozic acid is squalene synthase [31], while azoles target the C-14 sterol demethylase [32]. Squalene synthase and the C-14 sterol demethylase are encoded by ERG9 and ERG11, respectively. The results presented in Table 1 show that

Table 1 Expression of the ERG9-lacZ fusion in a wild-type strain in response to ergosterol biosynthetic inhibitors L-Galactosidase activity Inhibitor

WM

ERG9-lacZ (pIU850)

Ratio of treated/ non-treated

None Zaragozic acid A Ketoconazole

^ 60 10

97 þ 4 513 þ 18 197 þ 20

^ 5.3 2.0

Quantitation of L-galactosidase enzyme activity from the ERG9 promoter-lacZ fusion was carried out as described in Section 2. L-Galactosidase activity with S.D. represent nmol ONPG hydrolyzed per min per mg protein and is the average of values obtained from two independent transformants assayed in duplicated over three days. BWG1-7a is a wild-type (wt) strain.

treatment of wild-type cells with zaragozic acid A increased ERG9 expression 5.4-fold and ketoconazole increased ERG9 expression two-fold. HMG-CoA reductase catalyzes an early step in ergosterol biosynthesis and is located upstream of ERG9 (Fig. 1). In yeast there are two isoforms of HMG-CoA reductase [11]. Robinson et al. [4] demonstrated that treatment of wild-type cells with lovastatin, an inhibitor of HMG-CoA reductase, resulted in a 5^6-fold increase in squalene synthase mRNA levels. Table 2 shows that a mutation in the HMG1 gene encoding the major isoform of HMG-CoA reductase resulted in a two-fold increase in ERG9 expression while a mutation in the HMG2 gene, the minor isoform, resulted in no change in ERG9 expression. The di¡erences in expression observed by Robinson and this study can be explained by: (1) treatment with lovastatin blocks both isoforms, and (2) their study measured both expression and mRMA stability. Thorsness et al. [33] and Parks et al. [34] have suggested that during late stationary phase, the HMG2 gene product was the major isoform. The data in Table 2 demonstrate that expression of ERG9 in hmg1 and hmg2 was identical in log and late stationary phase. Arthington-Skaggs et al. [9] have established that hmg1 and hmg2 mutants show a 50 and 20% decrease, respectively, in total end-product ergosterol. While this decrease in end product sterol is not su¤cient to result in an ergosterol requiring phenotype, it is su¤cient to signal for an increase in ERG9 expression in an hmg1 mutant but not an hmg2 mutant.

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Arthington-Skaggs et al. [9] and Smith et al. [35] have demonstrated that a lesion in the ergosterol biosynthetic pathway results in increased ERG3 expression. In addition, they presented evidence that lesions in sterol biosynthetic genes e¡ect ERG3 expression di¡erently. Table 2 illustrates that mutations in the ergosterol biosynthetic pathway e¡ect ERG9 expression and that most of the ergosterol mutations result in di¡erent levels of induction. This suggests that the cell is capable of modulating the degree of ERG9 expression in response to the accumulation of speci¢c sterol intermediates. Mutations in erg2 and erg3 both resulted in signi¢cant 2.4-fold increases in ERG9 expression while a mutation in erg4 resulted in a modest but not signi¢cant 1.4-fold increase. A mutation in erg5 resulted in a modest 1.7-fold decrease and a mutation in erg6 resulted in no change in ERG9 expression. The erg2, erg3, erg4, erg5, and erg6 mutations in S. cerevisiae are non-auxotrophic for sterols and result in the accumulation of the sterol intermediates: ergosta-5,8,22-trien-3L-ol and ergosta-8-en-3L-ol for erg2, ergosta-7,22-dien-3L-ol for

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erg3, 24(28)-dehydroergosterol for erg4, ergosta-5,7dien-3L-ol, ergosta-7,24(28)-dien-3L-ol and zymosterol for erg5, and cholesta-8,24-dien-3L-ol and cholesta-5,7,22,24-tetraen-3L-ol for erg6. Intracellular sterols can exist in two forms, either as free sterols localized to the membrane or as esters conjugated to long chain fatty acids and sequestered to lipid storage droplets. Esteri¢cation is mediated by acyl-CoA:cholesterol acyltransferase (ACAT) enzymes in mammals [36]. Yeast have two ACAT related enzymes, ARE1 and ARE2, which catalyze esteri¢cation of intracellular sterols and recent reports have indicated that the inability to esterify intracellular sterols down-regulates the ergosterol biosynthetic pathway. Yang et al. [37] have demonstrated that incorporation of [14 C] acetate into non-saponi¢able lipids down-regulated sterol biosynthesis 2^3fold in esteri¢cation de¢cient strains. Table 2 demonstrates in an are1are2 double mutant, ERG9 expression is unchanged, indicating that ERG9 is not a target of esteri¢cation down-regulation as proposed by Yang et al.

Table 2 Expression of the ERG9-lacZ fusion gene in an ergosterol biosynthetic mutant background Strain (genotype)a

L-Galactosidase activity ERG9-lacZ (pIU850)

JRY527 (wt) JRY1159 (hmg1) JRY1160 (hmg2)

BWG 1-7a (wt) erg6 erg2 erg3 erg5 erg4 ARE1, ARE2 are1, are2 ARE1, are2 are1, ARE2

Ratio of mutant/wt ^

GCN4-lacZb

Ratio of mutant/wt

58 þ 11

^

102 þ 10 (100 þ 6)c 184 þ 24 (190 þ 15) 126 þ 23 (108 þ 13)

1.8 (1.9)c 1.2 (1.1)

56 þ 5

1.0

58 þ 9

1.0

104 þ 7 80 þ 3 252 þ 44 245 þ 47 60 þ 12 143 þ 22

^ 0.8 2.4 2.4 0.6 1.4

28 þ 4 32 þ 5 27 þ 4 31 þ 6 35 þ 6 29 þ 5

^ 1.1 1.0 1.1 1.3 1.0

90 þ 8 95 þ 10 80 þ 9 90 þ 5

^ 1.1 0.9 1.0

35 þ 6 33 þ 3 38 þ 4 32 þ 3

^ 0.9 1.1 0.9

Quantitation of L-galactosidase enzyme activity was carried out as described in Section 2 (see legend to Table 1). Each category of mutants includes an isogenic wild-type (wt) strain as a control. b GCN4 promoter-lacZ fusion serves as a control to verify that the regulatory e¡ect is speci¢c to ERG9. c Assays in parentheses were performed at late stationary phase (48 h at 30³C). a

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Table 3 Characterization of mutants that increased ERG9-lacZ fusion gene expression Strain MKY2A

Spec. act. 24 þ 4

Ratio of mutant/wt

Nystatin phenotypea

Accumulated sterolsb

^

2.5

ergosterol (68), episterol (10), zymosterol (10), fecosterol (5), lanosterol (4) mono oxygenated squalene (41), di-oxygenated squalene (34), ergosterol (10), lanosterol (4) ergosta-5,7-dien-3L-ol (85), lanosterol (15) ergosta-8,14,24(28)-trien-3L-ol (32), 4-methyl-ergosta8,14,24(28)-trien-3L-ol (24), ergosterol (24), lanosterol (8) mono oxygenated squalene (62), di-oxygenated squalene (29), ergosterol (2) ergosta-7,22-dien-3L-ol (55), ergosta-7-en-3L-ol (21), fecosterol (13) ergosta-7,22-dien-3L-ol (39), ergosta-7-en-3L-ol (26), fecosterol (18) fecosterol (66), episterol (15), lanosterol (10), zymosterol (5) ergosterol (72), zymosterol (9), episterol (5), fecosterol (4), lanosterol (4)

MKY9

127 þ 10

5.3

7.5

MKY10 MKY11

60 þ 5 80 þ 6

2.5 3.3

5.0 5.0

MKY12

137 þ 15

5.7

7.5

MKY13

77 þ 8

3.2

5.0

MKY14

60 þ 6

2.5

5.0

MKY15 MKY16

90 þ 7 144 þ 12

3.8 6.0

5.0 2.5

Quantitation of L-galactosidase enzyme activity was carried out as described in Section 2 (see legend to Table 1). The lowest concentration of nystatin (Wg/ml) that supported growth after 48 h at 30³C. b The major sterols accumulating in each strain are listed with percentage of each sterol in parentheses. a

3.2. A genetic screen to isolate mutants that increase expression of ERG9 In order to isolate mutants that cause an increase in ERG9 expression a strain with two di¡erent integrated ERG9 promoter fusions was constructed. This strain, MKY2A, contained a lacZ and HIS3 fusion allowing for both L-galactosidase assays for the lacZ fusion and resistance to the histidine biosynthetic inhibitor 3-amino-1,2,4-triazole (3-AT) for the HIS3 fusion. After UV mutagenesis, 10 000 colonies were screened for resistance to normally inhibitory levels of 3-AT (2 mM). Eight mutants were obtained that were resistant to at least 10 mM of 3-AT and had a signi¢cant 2.5-fold or greater increase in ERG9 expression (Table 3). These eight were further characterized as to their sterol pro¢le. All eight were tested for resistance to nystatin, a polyene inhibitor. Ergosterol mutants are resistant due to the absence of ergosterol in their plasma membrane [38]. Seven of the eight showed resistance to nystatin while one, MKY16, was similar to wild-type (Table 3). This indicated that the seven others were most likely ergosterol biosynthetic mutants and their sterol content was analyzed by gas chromatography mass spectrometry (GC/MS). The accumulated sterols for

each mutant are listed in Table 3. Strains MKY9 and MKY12 had a 5.3- and 5.7-fold increase in ERG9 expression respectively, the largest fold induction observed in this study. Both strains grew on media containing 20 mM 3-AT, and accumulated 75 and 91% mono- and di-oxygenated squalene respectively, as indicated by GC/MS. This sterol accumulation pro¢le is phenotypically identical to erg7 [39]. ERG7 is an essential gene that encodes the lanosterol synthase (squalene epoxide cyclase) and catalyzes the formation of lanosterol from squalene epoxide (Fig. 1). MKY9 and MKY12 mutants clearly exhibit the phenotype of a leaky erg7, indicating that mutations which alter intracellular sterol levels e¡ect ERG9 expression. Strains MKY13 and MKY14 had a 3.2- and 2.5-fold increase in ERG9 expression respectively, grew on 10 mM of 3-AT, and accumulated 55 and 39% ergosta-7,22-dien-3L-ol and 21 and 26% ergosta-7-en-3L-ol, respectively, as shown by GC/MS. This sterol accumulation pro¢le is identical to erg3 [40]. We previously demonstrated that ERG9 expression increased 2.4-fold in an erg3 deletion mutant (Table 2). Here two mutants (MKY13 and MKY14), de¢cient in C-5 desaturation, were isolated in the genetic screen and increased ERG9 expression 2.5^3.2-fold (Table 3). Strain MKY11

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had a 3.3-fold increase in ERG9 expression, grew on 10 mM 3-AT and accumulated 32% ergosta 8,14,24(28)-trien-3L-ol and 24% 4-methyl-ergosta8,14,24(28)-trien-3L-ol, as indicated by GC/MS. This sterol accumulation pro¢le is similar to erg24 [41] which is de¢cient in C-14 sterol reductase activity. MKY11 exhibits the phenotype of a leaky erg24. Strain MKY15 had a 3.8-fold increase in ERG9 expression, grew on 10 mM 3-AT, was nystatin resistant and accumulated 66% fecosterol, 15% episterol, and 10% lanosterol, clearly exhibiting an altered sterol phenotype. The sterol accumulation pro¢le for MKY15 has not been observed previously. Another mutant strain, MKY10, had a 2.5-fold increase in ERG9 expression, grew on 10 mM 3-AT and accumulated 85% ergosta-5,7-dien-3L-ol and 15% lanosterol. The accumulation of these two sterol intermediates was consistent with a heme mutation since heme de¢cient strains accumulate precursors of the cytochrome P450 enzymes [42]. To test whether MKY10 was a heme mutant, hemin was added to the media [43]. A wild-type sterol pro¢le of 65% ergosterol, 12% episterol, 12% zymosterol, 4% fecosterol and 3% lanosterol was obtained for MKY10 grown

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in the presence of hemin while L-galactosidase speci¢c activity was reduced to the wild-type value of 25 þ 4. Nystatin and 3-AT resistance were also reduced to wild-type levels of 2.5 Wg/ml and 2 mM respectively (data not shown). Finally, one mutant strain, MKY16, was identi¢ed in which ERG9 expression was increased six-fold, grew on 20 mM 3-AT but had a wild-type sterol pro¢le of 72% ergosterol, 9% zymosterol, 5% episterol, 4% fecosterol and 4% lanosterol and was not resistant to nystatin. Quantitative sterol analysis indicated that MKY16 and the wild-type MKY2A had 1.2 and 0.95% total sterol per mg of cell mass respectively, demonstrating that MKY16 is not a sterol mutant. 3.3. E¡ects of heme and oxygen on ERG9 expression Heme biosynthesis plays a critical role in the biosynthesis of ergosterol. Heme is a cofactor for two ergosterol hemoproteins encoded by ERG11 and ERG5, both of which are cytochrome P450 enzymes (see [1] for review). Heme is also associated as a cytochrome b5 cofactor with two enzymes, the C-5

Table 4 E¡ect of heme and oxygen on the expression of the ERG9-lacZ fusion gene L-Galactosidase activity Strain (genotype) TKY22 (hem1v) +50 Wg/ml N-ala TKY22 (hem1v) +0.5 Wg/ml N-ala BWG 1-7a (wt) +Oa2 BWG 1-7a (wt) 3O2 BWG 1-7a (wt) LPY22 (hap1) LWG1 (hap2) SHY40 (hap3)

RZ53-6 (wt) RZ53-6rox1 (rox1v)

ERG9-lacZ (pIU850)

Ratio of mutant/wt

114 þ 13 307 þ 39

^ 2.7

99 þ 13 48 þ 7

^ 0.5

128 þ 18 (131 þ 19)b 62 þ 8 (63 þ 12) 108 þ 8 (42 þ 9) 114 þ 9 (41 þ 10) 32 þ 2 93 þ 6

^ 0.48 (0.48)b 0.84 (0.32) 0.89 (0.31) ^ 2.9

Quantitation of L-galactosidase enzyme activity from the ERG9 promoter-lacZ fusion was carried out as described in Section 2 (see legend to Table 1). a Cells were grown with oxygen to early log phase then shifted to anaerobic growth under nitrogen for 4 h as described in Section 2. b Values in parentheses represent assays performed on cells grown on galactose.

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Fig. 2. The ERG9 promoter of Saccharomyces cerevisiae. The putative cis-acting elements of HAP1, HAP 2/3/4 (Heme Activator Protein), YAP1 (Yeast Activator Protein), and INO2/4 (E-box) are indicated.

sterol desaturase and the C-24 sterol methyl oxidase encoded by ERG3 and ERG25 respectively [1]. Thus, mutations in heme biosynthesis interfere with four steps in the ergosterol biosynthetic pathway. The role of heme in the regulation of ERG9 expression was explored by taking advantage of a hem1 mutant. HEM1 encodes N-aminolevulinic acid (N-ala) synthase, the ¢rst enzymatic step in this pathway. Rine et al. [33] have demonstrated that the addition of 0.5 Wg/ml N-ala allows for slow growth but gives an altered sterol pro¢le with minimal levels of ergosterol. However, the addition of 50 Wg/ml N-ala results in normal growth and a wild-type sterol pro¢le [33,44]. Table 4 shows that a hem1 mutant increased ERG9 expression three-fold (consistent with the 2.5fold increase previously shown for the leaky heme mutant MKY10). Sterols are not synthesized during anaerobic growth since molecular oxygen is required for squalene epoxidation and heme biosynthesis[45]. M'baya et al. [3] have demonstrated that squalene synthase activity is decreased during semi-anaerobic growth. Table 4 presents evidence that ERG9 transcription decreased two-fold when cells were shifted from aerobic to anaerobic growth. The data suggest that

e¡ect seen by M'baya et al. is occurring at the level of transcription. Because expression of ERG9 is sensitive to the availability of heme and oxygen, the e¡ect of three trans-acting regulatory systems previously demonstrated to regulate the expression of genes in response to heme and oxygen levels were studied. HAP1, HAP2/3/4, and ROX1 transcriptionally regulate genes primarily involved in oxygen requiring processes (see [46] for review), such as cytochrome synthesis (CYC1, CYC7, CYT1, CYB2, COR2 and COX4), sterol biosynthesis (HMG1 and ERG11), heme biosynthesis (HEM1 and HEM13), and fatty acid biosynthesis (OLE1). HAP1 (heme activated protein 1) is a transcriptional activator, uses heme as a cofactor and requires molecular oxygen for activity [47]. The putative HAP1 cis-element has 11/12 base pair homology to the consensus sequence 5PCGGNNNTANCGG-3P and is located at position 3168 to 3157 upstream of the ATG start codon (Fig. 2). To determine whether HAP1 regulates the expression of ERG9, the ERG9 promoter-lacZ fusion gene was used to measure ERG9 expression in the absence of the HAP1 activator. Table 4 shows that ERG9 expression decreased two-fold in a hap1 mu-

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tant background in either glucose or galactose (a partially non-fermentable carbon source). HAP2/3/4 (heme activated protein 2/3/4) is a heterotrimeric complex that functions as a transcriptional activator that uses heme as a cofactor and requires a non-fermentable carbon source for activity [48]. Hap2p and Hap3p contain DNA binding domains [49], and Hap4p contains the activation domain [50]. Two putative HAP2/3/4 cis-elements can be found in the ERG9 promoter. One has 4/5 base pair homology to the consensus sequence 5PCCAAT-3P and is located at position 3189 to 3185 and the other has 5/5 base pair homology to the consensus sequence and is located at position 3401 to 3397 upstream of the ATG start codon (Fig. 2). To determine whether HAP2/3/4 regulates the expression of ERG9, the ERG9 promoter-lacZ fusion gene was used to measure ERG9 expression in the absence of the HAP2/3/4 activator. In a hap2 or hap3 mutant, we measured a three-fold reduction in ERG9 expression when using galactose as a carbon source and no change in ERG9 expression when glucose was used as a carbon source (Table 4). ROX1 is a heme-induced repressor of hypoxic genes in yeast [51]. Target genes of Rox1p are repressed by an oxygen-dependent mechanism. When cells are grown aerobically, heme levels are su¤cient to induce high levels of Rox1p by HAP1 trans-activation, and hypoxic genes are repressed. As cells become limiting for oxygen, heme levels diminish, leading to a decrease in Rox1p repressor levels, derepressing hypoxic genes. To determine whether ROX1 regulates the expression of ERG9, the ERG9 promoter-lacZ fusion gene was used to measure ERG9 expression in the absence of the ROX1 activator. In Table 3 the data shows that ERG9 expression increased three-fold in a rox1 mutant. Rox1p binds to the 12 base pair hypoxic consensus sequence 5PYYYATTGTTCTC-3P [52]. Sequence inspection of the promoter of ERG9 revealed no cis-element for ROX1 (Fig. 2), suggesting that the ROX1 protein is not binding to the ERG9 promoter. It is possible that derepression of hypoxic genes during aerobic growth may alter normal gene expression of heme or ergosterol biosynthesis and that the increased ERG9 expression is a result of this perturbation. Since squalene synthase does not require heme or oxygen for enzymatic function, these data suggest that ERG9

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expression is indirectly regulated by heme and oxygen levels. 3.4. Identi¢cation of two trans-acting factors involved in membrane biogenesis and stress response that a¡ect ERG9 expression Two other putative cis-elements were identi¢ed by sequence inspection of the 756 base pair ERG9 promoter. The ¢rst element identi¢ed was a putative Ebox, 5P-CANNTG-3P, was identi¢ed at position 3590 to 3585 in the ERG9 promoter (Fig. 2) and has a 6/6 base pair homology to the consensus sequence. E-box elements serve as a recognition site for DNA-binding proteins of the basic helix-loop-helix (bHLH) family [53]. The E-box motif is the core of the inositol/choline responsive element (ICRE). Ashburner and Lopes [54] have described the ICRE consensus sequence as 5P-CATGTGAAAT-3P. Bachhawat et al. [55] have demonstrated that the canonical E-box is the binding site for the INO2/4 heterodimeric trans-activator complex. INO2/4 is a heterodimeric basic helix-loop-helix transcription factor that activates phospholipid and fatty acid genes. The complex is repressed in media containing inositol and choline by the repressor OPI1. In media free of inositol and choline, OPI1 is inactive and the INO2/4 complex is derepressed, thereby activating transcription of target genes. To determine whether INO2/4 regulates the expression of ERG9, the ERG9 promoter-lacZ fusion gene was used to measure ERG9 expression in the absence of the INO2/4 activator under repressed (with inositol and choline) and derepressed (without inositol and choline) conditions. Table 5 demonstrates that in either an ino2 or ino4 mutant, ERG9 expression increased 2.5^2.9-fold under derepressed conditions but was unchanged in the repressed state suggesting that INO2/4 acted as a repressor and not an activator. Consistent with this result is that an opi1 mutant had no e¡ect on ERG9 expression under repressed or derepressed conditions. ERG9 expression was also measured in a wild-type strain under these conditions and expression was 2.8-fold higher in the repressed state, indicative of INO2/4 repressor activity. This e¡ect is most likely not mediated by direct binding and repression of ERG9 expression but through an interaction between the phospholipid and ergosterol bio-

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Table 5 Expression of the ERG9-lacZ fusion gene in inositol regulatory mutants L-Galactosidase activity Strain (genotype)

Repressed/derepresseda

ERG9-lacZ (pIU850)

Ratio of mutant/wt

SH2 SH3 SH7 SH4

(wt) (ino2) (ino4) (opi1)

R R R R

64 þ 8 69 þ 10 63 þ 5 32 þ 6

^ 1.1 1.0 0.5

SH2 SH3 SH7 SH4

(wt) (ino2) (ino4) (opi1I)

D D D D

27 þ 3 67 þ 8 77 þ 8 30 þ 4

^ 2.5 2.9 1.1

BWG 1-7a (wt) BWG 1-7a (wt)

R D

98 þ 7 35 þ 5

^ 0.36

Quantitation of L-galactosidase enzyme activity from the ERG9 promoter-lacZ fusion was carried out as described in Section 2 (see legend to Table 1). a Repressed (R); cells grown in the presence of inositol and choline. Derepressed (D); cells grown in the absence of inositol and choline.

synthetic pathways based on the following evidence: (1) the consensus sequence in the promoter of ERG9 is just the core E-box and not the whole ICRE consensus sequence; (2) there are usually two to six ICRE elements in a target gene [56]; and (3), Robinson et al. [4] have previously shown that 3400 base pairs to the ATG will give full squalene synthase activity but the putative E-box is approximately 600 base pairs upstream of the start codon. The second element identi¢ed in the promoter of ERG9 was a cis-element for the yeast activator protein transcription factor yAP-1, located at position 3266 to 3260 of the ERG9 promoter (Fig. 2) and has a 7/7 base pair homology to the consensus sequence 5P-TGACTCA-3P. yAP-1 is a basic leucine zipper (bZIP) homodimer transcriptional activator that is responsive to cellular stress including oxidative stress and drug resistance [13]. Recently, a family of 8 YAP proteins have been reported [57] that have diverse biological functions related to cellular stress. yAP-1 is related to the mammalian Jun family of bZIP transcription activators which bind to the AP1 site (5P-TGACTCA-3P) which includes Gcn4p [58] in yeast. In a yap-1 mutant background, ERG9 expression decreased 2.3-fold (from 40 þ 8 in wildtype and 17 þ 3 in a yap-1 mutant), but in a gcn4v mutant background, ERG9 expression was unchanged (30 þ 2 in a wild-type and 28 þ 5 in a gcn4v mutant), indicating that yAP-1 is a positive

activator of ERG9 expression. Cellular stresses and a variety of drugs can severely impair cell function. To overcome this, yAP-1 targets many genes including ERG9, thereby increasing the level of free sterol in the cell making the plasma membrane more impermeable. In summary, this study has demonstrated that ERG9 expression is increased in response to a lesion in the ergosterol biosynthetic pathway (hmg1, erg2, erg3, erg9 and erg11), or the heme biosynthetic pathway (hem1), in which end-product sterol is not made. The data have shown that the genetic screen employed isolated mutants with phenotypes similar with ergosterol (erg7, erg24, erg3) and heme mutant phenotypes. One mutant with a non-ergosterol phenotype was also isolated. Mutations which result in the accumulation of squalene like sterol intermediates have the greatest e¡ect on ERG9 transcription and mutations which result in the accumulation of ergosterol like intermediates have the smallest e¡ect on ERG9 transcription. In addition, ERG9 is activated at the level of transcription by HAP1, HAP2/ 3/4, ROX1 and yAP-1 and repressed by INO2/4 demonstrating that ERG9 expression is also responsive to heme, oxygen, stress, and phospholipid/fatty acid biosynthesis. These results provide new insights into the regulation of squalene synthase in S. cerevisiae and pave the way for further investigations in both yeast and mammals.

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Acknowledgements We wish to thank all of the individuals previously mentioned for the gift of strains and plasmids. We also thank M. Kurtz (Merck) for the gift of zaragozic acid. This work was supported in part by a Predoctoral Fellowship from the American Heart Association (Indiana A¤liate) to M.A.K. and a National Institute of Health Grant 1R01 AI38598 to M.B. M.A.K. would also like to thank Daniel Gachotte for numerous helpful discussions.

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