Positive and negative regulation of squalene synthase (ERG9), an ergosterol biosynthetic gene, in Saccharomyces cerevisiae

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Positive and negative regulation of squalene synthase (ERG9), an ergosterol biosynthetic gene, in Saccharomyces cerevisiae Matthew A. Kennedy 1 , Martin Bard * Department of Biology, Indiana University Purdue University, Indianapolis, 723 West Michigan Street, Indianapolis, IN 46202, USA Received 7 June 2000 ; received in revised form 26 September 2000; accepted 28 September 2000

Abstract To identify regulatory cis-elements in the proximal promoter of the yeast ERG9 squalene synthase gene, promoter deletion analysis was performed. This approach identified two regulatory elements, one an upstream repressing cis-element (URS), and the other an upstream activating cis-element (UAS). Electromobility shift assays (EMSAs) demonstrated that distinct proteins bind each element. Genetic screens were performed to identify yeast mutants that altered expression of ERG9 promoter^reporter gene fusions. Three non-ergosterol biosynthetic pathway genes were identified. A mutation in TPO1(YLL028W) led to a 5.5-fold increase in ERG9 expression while mutations in YER064C and SLK19 (YOR195W) led to a 3.1- and 5.6-fold decrease, respectively. Deletion analysis of these genes demonstrated that TPO1 and SLK19 specifically regulated ERG9 expression when tested with several different promoter^reporter gene fusions. Additionally, EMSAs demonstrated that extracts derived from the TPO1 deletion strain was unable to shift the repressing cis-element while protein extracts from the SLK19 deletion strain had a reduced shift of the activating cis-element. Furthermore, these two mutants showed quantitative differences in sterols and antifungal drug susceptibilities consistent with their role in regulating ERG9 expression. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Ergosterol ; Squalene synthase; Transcription; Regulation ; Promoter; Saccharomyces cerevisiae

1. Introduction It is well established that sterol levels are properly maintained in animal cells through a transcriptional feedback regulatory mechanism in which excess cellular sterol represses gene expression [1,2]. Saccharomyces cerevisiae also maintain proper ergosterol levels through a transcriptional feedback regulatory mechanism [3^10]. Even though this mechanism has been observed by several investigators, the molecular details governing this regulation is unknown. We have previously shown that squalene synthase

Abbreviations : 3-AT, 3-amino-1,2,4-triazole; EMSA, electromobility shift assay; PCR, polymerase chain reaction ; DEPC, diethyl pyrocarbonate; UAS, upstream activating sequence; URS, upstream repressing sequence * Corresponding author. Fax: +1-317-274-2846; E-mail : [email protected] 1 Present address: Department of Cardiology, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA.

(ERG9) expression is regulated by mutations in the ergosterol biosynthetic and heme pathways [7]. Additionally, we demonstrated that the yeast heme activator proteins HAP1 and HAP2/3/4, the phospholipid activator INO2/ 4, and the stress activator YAP-1 play a role in the regulation of squalene synthase. HAP1 was shown to be involved in the regulation of HMG-CoA reductase (HMG1), another sterol biosynthetic gene [11]. In the present study we extend these analyses to delineate the cis-elements required for full expression and to identify genes that play a role in this regulation. We hypothesize the existence of several novel cis-elements in the promoter of the yeast squalene synthase gene that are required for proper regulation and expression. To identify such cis-elements, promoter deletion analysis and electromobility shift assays (EMSAs) were performed. Squalene synthase regulation and expression were measured using promoter^reporter gene fusion constructs with lacZ and HIS3. These approaches identi¢ed two novel cis-elements in the promoter of squalene synthase; one that positively regulates expression through an upstream activating sequence (UAS), and one that negatively regulates expression through an upstream repressing sequence (URS).

0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 2 4 6 - 3

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Current studies demonstrate that each element binds distinct protein(s). To identify new genes participating in the regulation of squalene synthase, yeast mutants were isolated in genetic screens and characterized. This approach identi¢ed YER064C, SLK19 (YOR195W) and TPO1 (YLL028W), as three non-ergosterol biosynthetic genes that participate in the regulation and expression of squalene synthase. Current studies demonstrate that two of these genes, TPO1 and SLK19, are under the conditions tested, speci¢c for squalene synthase expression. Null mutants were shown to have altered levels of total sterols and susceptibilities to antifungal drugs implying a role in both regulating squalene synthase expression and £ux through the ergosterol biosynthetic pathway. These ¢ndings show that squalene synthase is transcriptionally regulated both in a positive and negative manner. While the exact molecular mechanism of the transcriptional regulation of squalene synthase is not yet completely understood, data presented here further re¢ne the details of this extremely complex regulatory circuit. 2. Materials and methods 2.1. Strains, plasmids, media and genetic methods Yeast strains are described in Table 1. Plasmid p180, a GCN4^lacZ promoter fusion in YCp50 [12] was provided by R. Wek (Indiana School of Medicine, Indianapolis, IN, USA); pLG669-Z, a CYC1^lacZ promoter fusion in Yep24 [13] was provided by L. Guarente [14]; and pYLZ-6 and pYLZ-2 were provided by H. Hermann [15]. Yeast strains were grown at 30³C in 1% yeast extract, 2% peptone, 2% glucose (YPD) or complete synthetic me-

dium (0.67% yeast nitrogen base, 2% glucose; CSM) with appropriate nutrients omitted as required to maintain plasmid selection [16]. Sporulation media and tetrad dissection were performed by standard methods [17]. 3-Amino-1,2,4-triazole (3-AT) was used to select for yeast mutants expressing histidine [16]. 3-AT was added to appropriate CSM media to concentrations of 2, 5, 10 and 20 mM. 5-Fluoroorotic acid (5-FOA), a toxic pyrimidine analog, was used to negatively select for strains carrying URA3 based plasmids [18]. Yeast strains were transformed using the lithium acetate method [19]. Plasmids were isolated from yeast strains using the rapid isolation method [20]. Escherichia coli strains were transformed as described by Ausubel et al. [21] and grown at 37³C in Luria^Bertani (LB) media with 50 Wg/ml ampicillin. 2.2. Plasmid constructions The construction of the ERG9^lacZ reporter gene fusion plasmid pIU850 was previously described [7]. To make pIU850 an integrating ERG9^lacZ promoter fusion vector at the URA3 locus, the 1.7 kb ScaI CEN6, ARSH4 fragment of pIU850 was replaced with the 1.2 kb ScaI fragment of the integrating vector pRS306 [22]. Integration was accomplished by linearization with StuI in the URA3 locus, transformation and selection for uracil prototrophy. Overlapping 5P promoter deletions of ERG9^lacZ fusions were constructed similar to pIU850 described above except that the MB-MK-2 polymerase chain reaction (PCR) primer was replaced by 5P deletion primers, pIU850-320, pIU850-270, pIU850-220, pIU850-175 and pIU850-120 (Table 2). pIU850-379 was constructed by deleting a 377 bp EcoRI^XbaI fragment from pIU850, ¢lling in the ends and religating the vector.

Table 1 Yeast strains used in this study Strains (referencea )

Genotype

BWG1-7a [27] H1716 [7] H2060 [7] BY4741 [24] MKY2A [7] MKY16 [7] MKY55 MKY56 MKY26 MKY29 MKY30 MKY31 MKY34 MKY35 vyer064c vyor195w vyll02w

MATa, ade1^100, his4^519, leu2^3,-112, ura3^52 MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, ino1^13 MATK K, gcn4v, his3^609, leu2^3,-112, ura3^52, trp1v63 MATa, his3v1, leu2v0, met15v0, ura3v0 MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, ino1^13 [ERG9^lacZ, URA3] [ERG9^HIS3, LEU2]b MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, ino1^13, tpo1^1 [ERG9^lacZ, URA3] [ERG9^HIS3, LEU2] MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, trp1v63, tpo1^1 [ERG9^HIS3, LEU2] MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, trp1v63 [ERG9^HIS3, LEU2] MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, ino1^13 [ERG9^HIS3, LEU2]+pIU900 (ERG9^lacZ, URA3, 2 W) MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, ino1^13, slk19^1 [ERG9^HIS3, LEU2]+pIU900 (ERG9^lacZ, URA3, 2 Wm) MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, ino1^13, yer064c-1 [ERG9^HIS3, LEU2]+pIU900 (ERG9^lacZ, URA3, 2 Wm) MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, ino1^13, yer064c-2 [ERG9^HIS3, LEU2]+pIU900 (ERG9^lacZ, URA3, 2 Wm) MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, ino1^13, yer064c-3 [ERG9^HIS3, LEU2]+pIU900 (ERG9^lacZ, URA3, 2 Wm) MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, ino1^13, slk19-2 [ERG9^HIS3, LEU2]+pIU900 (ERG9^lacZ, URA3, 2 Wm) MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, ino1^13, vyer064c: :HIS3 MATa, gcn4v, his3^609, leu2^3,-112, ura3^52, ino1^13, vslk19: :HIS3 MATa, his3v1, leu2v0, met15v0, ura3v0, vtpo1: :KANMX4

a

Strains not referenced were constructed for this work. H1716 is the isogenic wild type of vyer064c and vslk19. BY4741 is the isogenic wild type of vtpo1. b The ERG9^lacZ fusion was integrated at the URA3 locus and the ERG9^HIS3 fusion was integrated at the LEU2 locus.

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Table 2 Oligonucleotides used in this study Name

Sequencea

Use in workc

MB-MK-1 MB-MK-2 pIU850-320 pIU850-270 pIU850-220 pIU850-175 pIU850-120 pYLZ6-1 pYLZ6-2 UAS-FWD UAS-REV URS-FWD URS-REV YER064C-FWD YER064C-REV YOR195W-FWD YOR195W-REV YER064C-CONF YOR195W-CONF MB-AS-5 YCP50-FWD YCP50-REV MKPE-1

5P-GGGGGGGATCCATTGTGTGTGTGTGATATGTGACGTG-3P 5P-GGGGGGAATTCGTACCGTCACAATGTAGGGCTATATAT-3P 5P-GGGGGGAATTCGCAGGAACTACAAACCTTGC-3P 5P-GGGGGGAATTCCTCTGACTCAGTACATTTCATAGCC-3P 5P-GGGGGGAATTCCCATCCTATTTGCTTTGCCC-3P 5P-GGGGGGAATTCTCGGAAGGCGTTATCGG-3P 5P-GGGGGGAATTCCCACGGGCTATATAAATGG-3P 5P-CAATACGCAAACCGCCTG-3P 5P-AGGCGATTAAGTTGGGTA-3P 5P-TACCGACTTACCATCCTATTTGCTTTGCCCTTTTTCTTTTCCACTGCACTTTGCATCGGAAGGCG-3P 5P-CGCCTTGGCATGCAAAGTGCAGTGGAAAAGAAAAAGGGCAAAGCAAATAGGATGGTAAGTCGGTA-3P 5P-CTCTAACTCCGCAGGAACTACAAACCTTGCTTACACAGAGTGAACCGCTGCCTGGCGTGCTCTGACTCA-3P 5P-TGAGTCAGAGCACGCCAGGCAGCAGGTTCACTCTGTGTAAGCAGGTTTGTAGTTCCTGCGGAGTTAGAG-3P 5P-GGAAAAATAAGCTATGATAAATAGTTCATAGTAATGCAGGTGGGGATGATGCAAGCGGCctggcgggtgtcggggctggc-3Pb 5P-CTCTAGCTGTATATTACCTTTAGCATATTCAAACATCTTAACCGGAAAATATCTCAgcttgccgatttcggcctattg-3P 5P-GCATCATTGGTGTCAAGGGGCACCCAGTTAAAAAAGGTTTTGAGCACATATCGTAATTctggcgggtgtcggggctggc-3P 5P-CTAACAACTTGAGTAACTCCTGCTTTTCTTCTCTTTCTGAAGACAGCTGCTTGTTCAATTgcttgccgatttcggcctattg-3P 5P-GCAGTTGTTTGTATATGAAGG-3P 5P-CGTACACTCTGCTGGCC-3P 5P-CCAGCCGGAATGCTTGGC-3P 5P-GCGATCATGGCGACCACACCC-3P 5P-GGTGATGCCGGCCACGATGCG-3P 5P-GCTGCCTTCATCTCGACCGG-3P

C C PD PD PD PD PD S S EMSA EMSA EMSA EMSA GD GD GD GD S S S S S PE

a

Underlined sequence corresponds to either the restriction site GGATCC (BamHI) or GAATTC (EcoRI). Lowercase sequence corresponds to the common pRS313 region outside of the HIS3 cassette. c C, cloning ; PD, promoter deletion; EMSA, electromobility shift assay; GD, gene disruption ; PE, primer extension analysis; S, sequencing. b

2.3. UV mutagenesis and cloning UV mutagenesis was performed on MKY2A and MKY26 by standard methods described by Rose et al. [23] to a survival of 20%. Mutants of MKY2A obtained from UV mutagenesis were selected for their ability to survive on inhibitory levels of 3-AT (10^20 mM) and had increased L-galactosidase activity while mutants of MKY26 were selected for their inability to survive on CSM-HIS þ 2 mM 3-AT and had decreased L-galactosidase activity. MKY55 was isolated as a segregant from a diploid made from a cross between MKY16 and H2060 (see Table 1), and used to clone the wild type allele with a YCp50 based library from Rose et al. [12] by selecting for loss of 3-AT resistance and a decrease in L-galactosidase activity. Plasmids were extracted from complementing clones and subcloned by standard methods [21] and sequenced with primers YCP50-FWD and YCP50-REV (Table 2). The corresponding deletion strain vyll028w and the isogenic wild type BY4741 were purchased from Research Genetics [24]. Mutants of MKY26 cleared of the high copy ERG9^ lacZ fusion were used to clone their wild type alleles with the YCp50 library by selecting for histidine prototrophy and wild type sensitivity to 3-AT. Plasmids were isolated and sequenced as above. Deletions of cloned mutants was accomplished by insertion of the HIS3 locus by

one-step PCR disruption of the complete open reading frame (ORF). PCR primers for one-step disruption consisted of 60 bp of £anking region to the ORF at the 5P end of the primer plus 20 bp of £anking region to the HIS3 locus in pRS313 at the 3P end of the primer. Primers YER064C-FWD/YER064C-REV and YOR195W-FWD/ YOR195W-REV were used to disrupt the wild type H1716 strain by this method (Table 2). Disruption was con¢rmed by PCR using a primer upstream of the FWD primers (YER064C-CONF and YOR195W-CONF), and MB-AS-5, an internal primer for the HIS3 gene (Table 2). 2.4. L-Galactosidase assays Quantitative liquid assays were done as described by Rose and Botstein [25], and the exact protocol used has been described [7]. Two non-ergosterol biosynthetic pathway promoter reporter fusions (GCN4^lacZ and CYC1^ lacZ) and a promoter-less lacZ vector were used as controls to verify that the regulatory e¡ect observed was speci¢c for ERG9 in the L-galactosidase assays. 2.5. EMSAs Yeast protein extracts used for the EMSA were described previously [26,27]. Total protein concentrations were determined by Bradford assay [28]. Synthesized oligonucleotides were used in the EMSA (Life Technologies).

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UAS-FWD and its reverse complement UAS-REV correspond to 3229 to 3165 bp of the ERG9 promoter while URS-FWD and its reverse complement URS-REV correspond to 3320 to 3260 bp (see Table 2). Each oligo (0.1 Wg), was labeled separately with [Q-32 P]ATP (3000 Ci/mmol, Amersham), annealed and puri¢ed by polyacrylamide gel electrophoresis (PAGE) and visualized by autoradiography. Gel shifts were performed with 20 Wg protein extract pre-incubated on ice for 10 min (20 mM HEPES (pH 7.9), 4 mM Tris^HCl (pH 8.0), 60 mM KCl, 0.2 mM EDTA, 12% glycerol) containing 1 Wg poly(dI-dC)Wpoly(dI-dC), (Pharmacia) in 20 Wl. Radioactively labeled probes (30 000 cpm, V0.2 ng) were added, and the samples incubated for 30 min at room temperature. To separate DNA^protein complexes, samples were loaded onto a 4% non-denaturing PAGE gel (pre-run for 30 min at 20 mA) in 0.5U Tris^borate^EDTA bu¡er. Electrophoresis was carried out at room temperature, 20 mA, for approximately 2.5 h. The gels were then transferred to Whatmann 3MM ¢lter paper, dried and autoradiographed. For EMSA competition assays, cold probes were made by annealing oligonucleotide primer pairs, purifying by UV shadow [21] and were then quanti¢ed. Cold probes were diluted from 0.05- to 10 000-fold labeled probe concentration (0.2 ng) and added immediately after the addition of labeled probes in the DNA^protein binding assays. The band intensity of shifted complexes was quanti¢ed by densitometry scanning with a Bio-Rad densitometer. 2.6. RNA isolation and primer extension analysis Total RNA was extracted from a 100 ml culture of BWG1-7a grown to 2U107 cells per ml in YPD. Cells were harvested at 4³C, washed 3U in 1/5 volume bu¡er (0.1 M Tris, pH 7.5, 0.1 M LiCl, 1 mM EDTA pH 7.5), and resuspended in a ¢nal volume of 1.5 ml. Diethyl pyrocarbonate (DEPC) treated glass beads (0.45^0.55 mm) were added and the cells were vortexed 6U20 s with 20 s intervals on ice. One percent sodium dodecyl sulfate was added to the bu¡er and 3U8 ml PCIA (25/24/1; phenol/ chloroform/isoamyl alcohol) was used to extract the RNA. The aqueous layer from the PCIA extractions were pooled and precipitated with 2.5 volumes of ethanol and resuspended in a ¢nal volume of 60 Wl of DEPC treated water. Poly A‡ mRNA was isolated using the PolyAT track kit (Promega). To map the site of transcription initiation of the ERG9 mRNA, primer extension analysis was performed using the primer extension system kit from Promega. Ten Wg of total RNA and 0.5 Wg of poly A‡ mRNA were hybridized to 2 ng of 32 P end-labeled primer MKPE-1 (Table 2), at 5.7U106 cpm/pmol. The extension reaction took place at 64³C for 30 min before initiating the extension. The reaction products were separated on an 8% polyacrylamide^urea sequencing gel along with a set of double-

stranded sequencing reactions of pIU850, using MKPE-1 as a primer in order to size the products, and visualized by autoradiography. 2.7. Sterol analyses Non-saponi¢able sterols were isolated as described previously by Molzahn and Woods [29]. 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 mU 0.25 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 a ¢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 [30] 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 pre-weighed 0.45 Wm nitrocellulose ¢lter. The cells were dried in a 75³C oven overnight and weighed. 2.8. Drug susceptibility analysis Drug susceptibilities of wild type and deletion mutant strains vyer064c, vyor195w, and vyll028w were conducted essentially as previously described [31], using cells harvested from overnight YPD plates grown at 30³C. Cells were suspended in YPD medium to a concentration of 107 cells per ml (optical density at 660 nm of V0.5). Cells were plated by transferring 2.5 Wl of the original suspension (100 ) plus 1031 and 1032 dilutions onto YPD plates containing the drug to be tested. The plates were incubated for 48 h at 30³C and observed for growth. Stock solutions of tolnaftate (Sigma), ketoconazole (ICN), and fenpropimorph (Crescent Chemical Co.) were prepared in dimethyl sulfoxide. Terbina¢ne (D. Kirsch, American Cyanamid, Princeton, NJ, USA) stock solution was prepared in ethanol, zaragozic acid A (M. Kurtz, Merck, Rahway, NJ, USA) was prepared in water, and nystatin (Sigma) was prepared in N,N-dimethyl formamide. 3. Results and discussion 3.1. ERG9 promoter deletion analyses In order to identify the cis-acting elements necessary for the observed regulated expression of ERG9, a series of ERG9 promoter deletions was created and fused to a lacZ reporter gene. L-Galactosidase enzyme activity generated from the deletion promoters transformed and inte-

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Fig. 1. Sequence of the ERG9 promoter of S. cerevisiae including the initiation codon (indicated in bold) and 756 bp upstream. URS and UAS cis-elements are underlined and coordinates are 3320 to 3270 and 3220 to 3175, respectively.

grated into the wild type strain BWG1-7a was compared to that of the wild type ERG9^lacZ reporter gene fusion as shown in Fig. 2. A full length construct of 756 bp (pIU850; Fig. 2A) exhibited the same amount of L-galactosidase activity as a 5P deletion of 377 bp (pIU850-379; Fig. 2B) and 436 bp (pIU850-320; Fig. 2C). This result indicated that 320 bp region of the promoter shown in Fig. 2C (pIU850-320) was su¤cient for ERG9 expression. Truncation by another 50 bp to a 270 bp promoter (pIU850-270) resulted in a two-fold increase in ERG9 expression (Fig. 2D), indicating that a URS is contained within the 50 bp of 3320 to 3270 bp (see Fig. 1 for sequence). A further truncation by 50 bp to a 220 bp promoter (Fig. 2E) showed the same two-fold increase in ERG9 expression indicating the absence of cis-elements in this region of the promoter. However, deletion of the next 45 bp leading to a promoter of 175 bp (pIU850-175) resulted in a four-fold drop in ERG9 expression (Fig. 2F), indicating that an UAS is located within the 45 bp of 3220 to 3175 bp (see Fig. 1 for sequence). Finally, a deletion of the next 50 bp to a promoter of 120 bp (pIU850-120) resulted in an additional 1.5-fold reduction in ERG9 expression (Fig. 2G). This suggests the possibility of a weak cis-element contained in this 50 bp region but was not investigated further in this study. pIU850-120,

which consists of the promoter from 10 bp upstream of the TATA box to the start codon (Fig. 2G), has 40% of the activity of the entire promoter pIU850-320 indicating a substantial amount of unregulated expression for ERG9 which may be due to chromatin remodeling or some other general control mechanism. A vector control, however, without an ERG9 promoter insert, had no speci¢c activity in the same wild type strain under the conditions tested, con¢rming that the residual expression seen in pIU850-120 is not an artifact of the reporter used. The results of the promoter deletion analyses (Fig. 2) indicate that there are two cis-elements, a URS and a UAS in the promoter of ERG9 required for normal expression. To map the transcription start site of the ERG9 mRNA and show that the cis-elements of the ERG9 promoter were proximal to the ERG9 mRNA transcript, primer extension was performed. One ERG9 mRNA transcript was observed (data not shown) which corresponds to the mRNA transcript starting at base 356 of the ERG9 promoter (indicated in Fig. 1). 3.2. Electromobility shift analysis of the UAS and URS promoter regions of ERG9 In order to show that protein(s) were actually binding at

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Fig. 2. Deletion analysis of the ERG9 promoter. A series of proximal deletions was constructed within the promoter region of the ERG9^lacZ reporter gene. On the left are schematic representations of the various constructs. Sequences corresponding to an UAS and URS, respectively, are indicated. A wild type strain (BWG1-7a) harboring these various integrated constructs were grown to late log phase and L-galactosidase assays were performed (shown on right). Speci¢c activity represent nmol ONPG hydrolyzed min31 mg31 protein and is the average of values obtained from two independent transformants assayed in duplicate over three days (S.D. þ 10^16%).

the UAS and URS regions in the promoter of ERG9, EMSAs were performed. Additionally, we wanted to determine if similar or di¡erent protein(s) bind to the UAS and URS. Double stranded oligonucleotide probes corresponding to the UAS (3230 to 3165) and URS (3330 to 3260) were made as described in Table 2 and are shown in Fig. 1. Ten bp of ERG9 promoter sequence was added to the ends of each probe to ensure that complete regulatory cis-elements would be represented. Each of the two probes (UAS and URS), gave a single major shifted complex in the presence of a wild type yeast protein extract (see arrow in Fig. 3A, lane 2 and Fig. 4A, lane 2, respectively). The labeled UAS probe could be competed o¡ with cold UAS probe (Fig. 3A, lanes 3^7), and

50% of the labeled UAS probe was lost with a 50-fold excess of cold UAS probe as determined by scanning densitometry. When cold URS probe was used in competition analysis with labeled UAS probe, the labeled UAS probe could not be competed o¡ with up to 1000-fold excess of cold URS probe (Fig. 3B, lanes 3^7). The labeled URS probe could be competed o¡ with cold URS probe (Fig. 4A, lanes 3^12), and 50% of the labeled URS probe was lost with a 250-fold excess of cold URS probe as determined by scanning densitometry. When cold UAS probe was used in competition analysis with labeled URS probe, the labeled URS probe could not be competed o¡ with up to 10 000-fold excess cold UAS probe (Fig. 4B, lanes 3^12). These results indicate that di¡erent

Table 3 Characterization of mutants identi¢ed in genetic screens that alter ERG9^lacZ gene expression Strain

CSM

MKY26 (wt) MKY29 MKY30 MKY31 MKY34 MKY35 MKY2A (wt) MKY16

+ + + + + + + +

CSM-HIS+3AT (mM)

Speci¢c activity

Ratio of mutant/wt

0

2

10

20

w/high copy ERG9^lacZ

w/integrated ERG9^lacZ

+ 3 + + + 3 + +

+ 3 3 3 3 3 + +

3 3 3 3 3 3 3 +

3 3 3 3 3 3 3 +

501 þ 22 117 þ 20 180 þ 10 215 þ 5 164 þ 20 119 þ 8 nd nd

26 þ 2 4.7 þ 0.8 8.3 þ 1.3 12 þ 2 8.3 þ 1 5.1 þ 0.5 25 þ 3 144 þ 8

Quanti¢cation of L-galactosidase enzyme activity is described in Section 2 (see legend to Table 2). a Mutations were cloned by complementation with a yeast genomic library and veri¢ed (see text).

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0.18 0.32 0.46 0.32 0.20 5.8

Mutationa wt slk19 yer064c yer064c yer064c slk19 wt tpo1

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Fig. 3. EMSA analyses of the UAS element in the promoter of ERG9 complexed with BWG1-7a wild type yeast protein extracts. (A) Labeled UAS probe was either complexed with extracts alone (lane 2) or in the presence of increasing concentrations of unlabeled UAS as a competitor (lanes 3^7). (B) Labeled UAS probe was either complexed with extracts alone (lane 2) or in the presence of increasing concentrations of unlabeled URS as a competitor (lanes 3^7).

protein(s) bind to the UAS and URS. Di¡erences in binding speci¢city could also be due to di¡erent binding a¤nities between the protein(s) and DNA or that one or more unknown proteins have becomes limiting. Alternatively, binding a¤nities may be a re£ection of the conditions of the EMSA. 3.3. Identi¢cation and characterization of mutants that increase ERG9 expression We hypothesized the existence of genes that regulate squalene synthase expression. To investigate this hypothesis, a genetic screen was previously performed in order to isolate mutants with increased ERG9 expression [7]. One mutant, MKY16, had a normal sterol content but ERG9 expression increased six-fold and grew on 20 mM 3-AT (eight-fold the normally inhibitory concentration of 2.5 mM 3-AT, Table 3). In this study we characterize the mutation responsible for the increased expression of ERG9 in MKY16. MKY16 was crossed to an isogenic wild type of opposite mating type (H2060). The resultant diploid had wild type L-galactosidase speci¢c activity (25 þ 3), and normal sensitivity to 3-AT (2.5 mM), indicating that the mutation was recessive. Sporulation of the diploid and dissection of tetrads was performed and the induced mutation segregated 2:2, indicative of a single gene. One segregant, MKY55 (Table 1), that contained the ERG9^HIS3 fusion and the induced mutation was used to clone the wild type allele. MKY55 was transformed with the URA3 based yeast genomic library [12]

and plated on CSM-HIS media. Transformants were scored for the ability to grow on 2 mM 3-AT but not 5^ 20 mM 3-AT. One transformant out of 3500 was obtained with a wild type 3-AT phenotype. Growth of this transformant on 5-FOA resulted in the loss of the plasmid and reversion to the mutant phenotype indicating that the wild type phenotype was due to complementation and not reversion or suppression. A plasmid with a 5 kb genomic insert was isolated from the clone and retransformed into MKY55 to verify complementation. Subsequent subcloning and sequencing identi¢ed TPO1 (YLL028W) as the complementing gene. It was formally possible that the cloned gene could show complementation of the HIS3 fusion but not the lacZ fusion. Therefore, an ERG9^ lacZ gene fusion construct was integrated into the trp1 locus of MKY55 and complementation by TPO1 was observed for both reporter fusions. When pIU850, a full length ERG9^lacZ reporter fusion was transformed into BY4741 and vtpo1, a 4.9-fold increase in ERG9 expression was observed in the mutant (Table 4), con¢rming that a mutation in TPO1 is responsible for the observed increase in ERG9 expression. TPO1 has been previously identi¢ed as a polyamine transporter [32] and a member of the drug:H[+] antiporter DHA12 family of multidrug e¥ux proteins in the major facilitator superfamily (MFS) [32]. Tpo1p is present on the vacuolar membrane [33]. In a twohybrid assay, Tpo1p interacts with Cup2p [34]. CUP2is a transcription factor that responds to cellular stress regulates iron uptake and like ERG9 is regulated by YAP-1 and HAP-1 [35].

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Fig. 4. EMSA analyses of the URS element in the promoter of ERG9 complexed with BWG1-7a wild type yeast protein extracts. (A) Labeled URS probe was either complexed with extracts alone (lane 2) or in the presence of increasing concentrations of unlabeled URS as a competitor (lanes 3^12). (B) Labeled URS probe was either complexed with extracts alone (lane 2) or in the presence of increasing concentrations of unlabeled UAS as a competitor (lanes 3^12).

3.4. Identi¢cation and characterization of mutants that decrease ERG9 expression A genetic screen was also performed in order to isolate mutants that have decreased ERG9 expression. The strain MKY26 (Table 1) which contains a high copy ERG9^lacZ construct and an integrated ERG9^HIS3 fusion allowed for scoring of both L-galactosidase speci¢c activity and resistance to 3-AT. After UV mutagenesis, ¢ve mutants out of approximately 7500 colonies had a greater than three-fold reduction in L-galactosidase activity. Two mutants, MKY29 and MKY35, could not grow on CSM-HIS and had a ¢ve-fold reduction in ERG9 expression (Table 3). Three mutants, MKY30, MKY31 and MKY34, could grow on CSM-HIS but not when supplemented with 3AT; and had a two- to three-fold reduction in L-galactosidase activity (Table 3). The high copy ERG9^lacZ plasmid was cleared and replaced by the integrating ERG9^ lacZ reporter fusion (integrated into the URA3 locus). The relative fold decrease in the speci¢c activities observed

with the integrated reporter are in good agreement with the high copy reporter fusion activities as shown in Table 3. To clone the wild type alleles of the induced mutations, MKY29 and MKY34 which had been cleared of the high copy ERG9^lacZ fusion construct were transformed with the URA3 based yeast genomic library and plated on CSM and CSM-HIS media and then transferred to CSM-HIS +0^20 mM 3-AT. Approximately 2000 transformants were tested for each of the two mutants. Seven transformants were isolated that complemented MKY29 and ¢ve that complemented MKY34. Growth of these transformants on 5-FOA resulted in the loss of the plasmid and reversion to the mutant phenotype. Plasmids were extracted and it was observed that upon retransformation, one plasmid complemented MKY29 and MKY35 while another plasmid complemented MKY30, MKY31, and MKY34. Subcloning and sequencing showed that the complementing gene for MKY29 and MKY35 was SLK19 (YOR195W, Table 3); and that the complementing gene for MKY30, MKY31, and MKY34 was YER064C (Table 3). YER064C

Table 4 Mutations that alter ERG9^lacZ expression are speci¢c for ERG9 Strain

ERG9^lacZ

H1716 (wt) vslk19 vyer064c BY4741 (wt) vtpo1

24 þ 3 4.2 þ 0.7 8.5 þ 1.4 40 þ 5 194 þ 13

Ratio of mutant/wt 0.18 0.35 4.9

GCN4^lacZa 26 þ 3 25 þ 3 26 þ 3 23 þ 3 26 þ 2

Ratio of mutant/wt 0.96 1.0 1.1

CYC1^lacZb 140 þ 15 145 þ 6 60 þ 9 180 þ 15 191 þ 20

Ratio of mutant/wt 1.0 0.43 1.1

Quanti¢cation of L-galactosidase enzyme activity is described in Section 2. L-galactosidase activity with S.D. represents nmol ONPG hydrolyzed. Values are reported as speci¢c activities (nmol mg31 min31 ) and are the average of two independent transformants assayed in duplicate over three days. a;b GCN4^lacZ (general control of amino acid biosynthesis), and CYC1^lacZ (iso-1-cytochrome c) reporter gene fusion constructs were used as a control to verify that the regulatory e¡ect is speci¢c to ERG9.

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and SLK19 genes were disrupted using PCR to completely replace the coding region for each gene with the HIS3 gene in the wild type strain H1716 and con¢rmed by PCR (data not shown). pIU850, the full length ERG9^ lacZ reporter fusion construct, was transformed into wild type H1716, vyer064c and vslk19. As shown in Table 4, vslk19 showed a 5-fold decrease in ERG9 expression while vyer064c showed a 3-fold decrease as compared to the isogenic wild type H1716. These results indicate that mutations in YER064C and SLK19 are responsible for the observed decrease in ERG9 expression. SLK19 was identi¢ed as a synthetic lethal with KAR3 [36]. A slk19 null is viable but contains long astral microtubules, short spindles, has a bypass of meiosis I with a partial mitotic arrest [36]. During anaphase, Slk19p-GFP localizes to the spindle midzone [36]. SLK19 may be a transcription factor as it contains several putative leucine zipper domains [36,37] and as such may have a role in the regulation of other pathways in addition to its role in karyogamy. YER064C is a gene of unknown function but is predicted to be a small molecule transporter localized to an integral membrane [38]. 3.5. Mutations that alter ERG9 expression are speci¢c for ERG9 To determine if the regulatory e¡ect is global or speci¢c for ERG9, three di¡erent promoter fusions were introduced into the deletion mutants and their isogenic wild types and L-galactosidase activity was measured. ERG9^ lacZ, GCN4^lacZ and CYC1^lacZ gene fusion constructs were introduced into H1716, vslk19, vyer064c, BY4741

185

and vtpo1. GCN4 is involved in the general control of amino acid biosynthesis [39] and CYC1 encodes the iso1-cytochrome c gene [40]. If the regulatory e¡ect observed is speci¢c for ERG9, then the speci¢c activities for the ERG9 fusion will change but not for the CYC1 or GCN4 fusions. The results shown in Table 4 demonstrate that vslk19 and vtpo1 are speci¢c for ERG9 since only the ERG9^lacZ fusion shows changes in L-gal speci¢c activity in these three deletion strains. However, vyer064c is not speci¢c for ERG9 since there is decreased activity of both the ERG9^lacZ and the CYC1^lacZ fusions in the vyer064c strain (Table 4), suggesting that YER064C has a more global regulatory role while SLK19 and TPO1 are speci¢c for their regulatory targets. 3.6. EMSAs of the UAS and URS in wild type and deletion strains that alter ERG9 expression In order to test whether SLK19, YER064C and TPO1 regulate ERG9 expression through the UAS or URS ciselements identi¢ed in the promoter deletion analysis above, EMSAs were performed with 20 Wg of protein extract from the deletion mutants and their isogenic wild types against labeled UAS and URS probes. Since vslk19 and vyer064c lead to decreased ERG9 expression, these gene products normally have a positive regulatory role and may act through the UAS. A 70% loss of the shifted complex between extracts of vslk19 and the UAS label probe is seen in lane 4 of Fig. 5A with no loss of protein^DNA complex between extracts of vslk19 and the URS (Fig. 5B, lane 4). This result is consistent with the idea that SLK19 has an activating role

Fig. 5. EMSA analyses of the UAS and URS elements in the promoter of ERG9 complexed with yeast null mutants or isogenic wild type protein extracts. (A) Labeled UAS probe was complexed with 20 Wg yeast cell extracts from wild type H1716 and BY4741 strains (lanes 2 and 5, respectively), and null mutant vyer064c, vslk19 or vtpo1 strains (lanes 3, 4 and 6, respectively). (B) Labeled URS probe was complexed with 20 Wg yeast cell extracts from wild type H1716 and BY4741 strains (lanes 2 and 5, respectively) and null mutant vyer064c, vslk19 or vtpo1 strains (lanes 3, 4 and 6, respectively).

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in ERG9 expression. This decrease, but not loss, of shifted complex suggests that the SLK19 gene product modulates the binding a¤nity or abundance of other protein(s) either directly or indirectly at the UAS. However, it is formally possible that a complex without SLK19 may still be able to bind with lowered a¤nity. Either explanation is plausible. YER064C, which regulates ERG9 expression in the same manner as SLK19, is not a partner of SLK19 since there is no change in the shifted complexes between extracts of vyer064c and the labeled UAS probe (Fig. 5A, lane 3) or URS probe (Fig. 5B, lane 3). It must be concluded that YER064C is not exerting its regulatory e¡ect through the UAS or URS. Perhaps a more global mechanism involving either the general transcription machinery or perhaps through one of the positive regulatory factors HAP1, HAP 2/3/4 or YAP-1 already shown to regulate ERG9 [7]. Since the vtpo1 strain leads to increased ERG9 expression, this gene product normally has a negative regulatory role and would possibly act through the URS. In lane 6 of Fig. 5B there is a complete loss of the shifted complex between extracts of vtpo1 and the labeled URS probe with no loss of shifted complex between extracts of vtpo1 and the UAS (Fig. 5A, lane 6). The data suggest that TPO1 exerts control over the binding of the factor(s) at the URS, consistent with the data from the ERG9 expression analyses described earlier. It remains to be established which protein(s) actually binds to the UAS and URS elements. SLK19 is a gene that contains a leucine zipper motif [37], which is found in a class of transcription factors. In addition it was identi¢ed in a synthetic lethal screen with mutants of Kar3p, a kinesin motor [36]. YER064C is a gene of unknown function with no known protein motifs [37]. TPO1 is a 12 membrane spanning protein similar to permeases and multidrug transporters [41] with high homology to the S. cerevisiae FLR1 gene, a £uconazole drug transporter [42,43] that is positively regulated by YAP-1. It is unlikely that a permease/transporter itself is regulating ERG9 expression at the level of binding to the URS cis-element but it is more likely that it is involved in a signaling cascade to

regulate expression perhaps through YAP-1 since it also regulates ERG9 expression. 3.7. Mutants that alter ERG9 expression have similar intracellular sterol content but have di¡erent sterol percentages of total cellular mass The non-ergosterol biosynthetic mutants isolated in the genetic screens that altered ERG9 expression all had a wild type sterol pro¢le and percentage of total sterol (0.9^ 1.2%). A typical sterol pro¢le of ergosterol (72%), zymosterol (9%), episterol (5%), fecosterol (4%), and lanosterol (4%) was observed. It is possible that while these point mutants regulate ERG9 expression at the molecular level, the mutations are too leaky to show a phenotypic perturbation in the sterol pathway. In order to determine if there was a discernible sterol phenotype for the mutants identi¢ed in this study, the intracellular sterol content was examined for null mutants of YER064C, SLK19 and TPO1 and their isogenic wild types by GC. The sterol pro¢les for vyer064c, vslk19 and vtpo1 strains were identical to their wild type strains. However, the vslk19 strain accumulated 0.47% of total sterol which was three-fold less than its isogenic wild type (1.27%). The vyer064c strain had total sterol that was similar to wild type (1.24 and 1.27%, respectively), suggesting that SLK19 has a speci¢c e¡ect on both ERG9 expression and ergosterol biosynthesis while YER064C perhaps has a more global and non-speci¢c e¡ect. The vtpo1 strain had 1.27% total sterol which represented a small 1.5-fold increase in total sterol over its isogenic wild type (0.85%), suggesting a speci¢c e¡ect on both ERG9 expression and ergosterol biosynthesis. 3.8. Mutants that alter ERG9 expression have altered susceptibilities to antifungal agents Mutants of the ergosterol pathway have altered susceptibilities to antifungal compounds. The three genes identi¢ed in this study have been shown to regulate expression of ERG9. Here we ask if null mutants of YER064C, SLK19 and TPO1 have a speci¢c a¡ect on the di¡erent

Table 5 Mutant strains of S. cerevisiae that alter ERG9 expression have altered susceptibilities to antifungal agents Drug Terbina¢ne Tolnaftate Zaragozic acid Ketoconazole Fenpropimorph Nystatin

Inhibitory concentration (Wg/ml)a H1716 (wt)

vslk19

vyer064c

BY4741(wt)

vtpo1

50 10 10 10 2.5 2.5

12.5 5 2.5 2.5 0.5 5

75 10 7.5 s 10 5 10

25 10 10 5 2.5 2.5

75 15 20 5 5 1.0

a

Concentration at which no growth appeared after 48 h. Cells were grown at 37³C to a density of 107 cells/ml and 2.5 Wl was inoculated at 100 , 1031 and 1032 dilutions.

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Fig. 6. A model of how altered transcriptional regulation of the ERG9 gene may e¡ect £ux through the ergosterol biosynthetic pathway under wild type (A) and mutant (B, C) conditions. Data that supports this model are discussed in the text.

enzymatic steps in the ergosterol pathway. The susceptibilities of the null mutants was measured as compared to isogenic wild type strains using various antifungal compounds that target di¡erent enzymes in the ergosterol biosynthetic pathway [44] as well as the end product ergosterol. Terbina¢ne is an allylamine antifungal inhibiting squalene epoxidase (ERG1). Tolnaftate is an antifungal inhibiting lanosterol synthase (ERG7), while zaragozic acid is a squalestatin antifungal inhibiting squalene synthase (ERG9). Ketoconazole is an azole antifungal that targets the sterol C-14 demethylase (ERG11), while fen-

propimorph, a morpholine antifungal, is an inhibitor of sterol v14-reductase (ERG24), and v8-v7 isomerase (ERG2). Finally, nystatin targets ergosterol located in the membrane. The determination of drug concentrations su¤cient to completely inhibit growth on nutrient media of vslk19, vyer064c, vtpo1, and their isogenic wild type strains are shown in Table 5. The concentration of nystatin required for complete growth inhibition of a wild type strain is 2.5 Wg/ml [29]. The vslk19 mutant is two-fold more resistant to nystatin than the wild type while the vyer064c mutant is four-fold more resistant. Lowering

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the £ux, the movement of sterol intermediates, through the pathway and subsequently making less ergosterol would explain the nystatin resistance for the vslk19 mutant but not for the vyer064c mutant since no change in total sterol was seen in the vyer064c strain. The vslk19 strain also had increased susceptibility to all of the antifungal drugs tested with the greatest increases in the susceptibility for fenpropimorph (¢ve-fold) and terbina¢ne, ketoconazole and zaragozic acid (all four-fold). The results suggest that lowering the £ux through the pathway is achieved by lowering the expression of many of the enzymes in the pathway making the vslk19 mutant more susceptible to antifungal drugs. This was not seen for the vyer064c strain which shows either no di¡erence in drug e¤cacy (tolnaftate and zaragozic acid) or a decreased susceptibility for terbina¢ne (1.5-fold), fenpropimorph (two-fold), and ketoconazole (1.5-fold). These results suggest that YERO64C and SLK19 regulate squalene synthase and the sterol pathway in di¡erent ways which may be due in part to their di¡erent speci¢city for the regulation of ERG9. The vtpo1 strain is 2.5-fold more sensitive to nystatin (Table 5) with either no change in susceptibility (ketoconazole), or a decrease in susceptibility for terbina¢ne (three-fold), tolnaftate (1.5-fold), zaragozic acid (twofold) and fenpropimorph (two-fold). The vtpo1 mutant accumulated more total sterol and as such may have more ergosterol in the membrane resulting in increased sensitivity to nystatin. It has not been determined however whether the excess sterol accumulates in the free or esteri¢ed sterol fraction. Furthermore, it is formally possible that changes in transport function itself may account for altered susceptibilities to the antifungal agents and not be related directly to changes in squalene synthase activity. However, the results here suggest that increasing the £ux through the pathway is achieved by stimulating the expression of several enzymes in the pathway making the vtpo1 mutant more susceptible to antifungal agents. The results lead us to a simple model for coordinated expression of ERG9 and sterol £ux. Under wild type logarithmic growth conditions, factors that regulate squalene synthase expression are in equilibrium and there is a steady state level of ERG9 expression and £ux through the sterol pathway (Fig. 6A). When conditions are such that the repressing factor that binds to the URS is ablated, expression of squalene synthase increases and that in turn increases the £ux through the pathway by increasing the intracellular concentration of enzymes in the pathway (Fig. 6B). This would explain the increase observed in total sterol, the sensitivity to nystatin and the decreased susceptibility to antifungal drugs. When conditions are such that the activating factor that binds to the UAS is reduced or ablated, expression of squalene synthase decreases, leading to a decrease in total sterol and lowered £ux (Fig. 6C). Concentrations of enzymes involved in sterol biosynthesis are lowered and thus have a greater susceptibility to antifungal agents.

At this time, several questions remain unanswered in this model. Is the sterol phenotype observed for each of these three mutants due to altered transcriptional regulation of squalene synthase alone? These studies cannot rule out post-transcriptional, translational and turnover mechanisms of regulation. Are several other sterol genes in the pathway coordinately regulated along with ERG9 or do changes in ERG9 expression alone account for the observed drug susceptibilities? Equally possible is that a sensor modulates the £ux in the pathway by monitoring levels of squalene synthase or farnesyl pyrophosphate pools and thus regulates £ux via an alternate pathway such as increased/decreased protein turnover. Further experiments will be needed to identify the answers to these questions. However, this study is important because it shows a correlation between ergosterol biosynthesis and ERG9 expression. In summary, this study has demonstrated that squalene synthase expression is regulated positively and negatively through two novel cis-elements and that the proteins that complex on these elements are di¡erent. Additionally, through genetic screens, two genes (SLK19 and YER064C) that play a positive role in regulated expression of ERG9, and one gene (TPO1) that plays a negative role were identi¢ed. Under the conditions tested, SLK19 and TPO1 have been shown to be speci¢c for regulating squalene synthase while YER064C has a more global role. Additionally, mutations in either SLK19 or TPO1 show marked or complete loss of protein:DNA complexes in EMSA analysis suggesting that the SLK19 gene product acts at the UAS and the TPO1 gene product acts at the URS. Finally, an altered sterol phenotype was observed in a wild type strain with a deletion for each of these genes and a simple model for the coordinated regulation of ERG9 expression and sterol £ux are discussed. In total, even though the nature of these interactions are not fully understood, the data presented in this study provide new insights into the molecular mechanisms of the complex regulatory circuit of squalene synthase in S. cerevisiae. Acknowledgements We wish to thank all of the individuals previously mentioned for the gift of strains, plasmids and reagents. We thank M. Kurtz (Merck) for the gift of zaragozic acid A and D. Kirsch (American Cyanamid) for the gift of terbina¢ne. We also thank S. Rhodes (IUPUI, Indianapolis, IN, USA), for the gifts of leupeptin and pepstatin A; and P. Crowell (IUPUI, Indianapolis, IN, USA) for the gift of the polyAT track mRNA isolation kit from Promega. This work was supported by a pre-doctoral fellowship from the American Heart Association, Midwest A¤liate to M.A.K. and National Institutes of Health Grants 1R01 AI38598 and R01 GM62104 to M.B.

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