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Mechanisms of Resistance

Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine-Sensitive Variants

Christoph Ruckenstuhl, Silvia Lang, Andrea Poschenel, Armin Eidenberger, Pravas Kumar Baral, Peter Kohút, Ivan Hapala, Karl Gruber, Friederike Turnowsky
Christoph Ruckenstuhl
1Institute of Molecular Biosciences, Karl-Franzens-Universität Graz, Graz, Austria
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Silvia Lang
1Institute of Molecular Biosciences, Karl-Franzens-Universität Graz, Graz, Austria
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Andrea Poschenel
1Institute of Molecular Biosciences, Karl-Franzens-Universität Graz, Graz, Austria
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Armin Eidenberger
1Institute of Molecular Biosciences, Karl-Franzens-Universität Graz, Graz, Austria
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Pravas Kumar Baral
2Institute of Chemistry, Karl-Franzens-Universität Graz, Graz, Austria
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Peter Kohút
3Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovak Republic
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Ivan Hapala
3Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovak Republic
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Karl Gruber
2Institute of Chemistry, Karl-Franzens-Universität Graz, Graz, Austria
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Friederike Turnowsky
1Institute of Molecular Biosciences, Karl-Franzens-Universität Graz, Graz, Austria
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  • For correspondence: friederike.turnowsky@uni-graz.at
DOI: 10.1128/AAC.00988-06
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  • FIG. 1.
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    FIG. 1.

    Growth of S. cerevisiae KLN1 strains carrying wild-type ERG1 (NS1) and various erg1 alleles on a centromere vector in the presence of terbinafine. Growth was monitored after 24 h of incubation at 30°C by measuring the OD600.

  • FIG. 2.
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    FIG. 2.

    Temperature sensitivities of mutants expressing Erg1 protein variants. Overnight cultures of KLN1 carrying the wild-type ERG1 gene (pNS1) and four erg1 alleles on a centromere vector were prepared at 30°C in YPD medium. The OD600 of the overnight cultures was adjusted to 0.1 (100), and 5 μl of each of the 100 to 10−3 dilutions was spotted on YPD agar plates and grown for 2 days at 30°C and 37°C.

  • FIG. 3.
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    FIG. 3.

    Expression of erg1 alleles: mRNA and protein levels of terbinafine-sensitive mutants. Cultures of wild-type and terbinafine-sensitive KLN1 strains were grown in YPD medium to early log phase. (A) Northern blot analysis. Total RNA was extracted from mechanically disrupted cells and 10 μg of total RNA was separated by agarose gel electrophoresis. Hybridization was performed as described in Materials and Methods, using an ERG1-specific DIG-labeled DNA probe. The amounts of the ACT1 mRNA and 25S rRNA were used to normalize the amount of RNA loaded on the gel. (B) Western blot analysis. Whole-cell extracts were prepared from 4 OD600 equivalents by NaOH disruption, followed by TCA precipitation. Precipitates were dissolved in 100 μl final sample buffer, and 5-μl aliquots were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis was performed with anti-Erg1p antibodies and anti-Sui2p antibodies as the loading control.

  • FIG. 4.
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    FIG. 4.

    Stability of Erg1p from S. cerevisiae KLN1(pNS1) carrying wild-type ERG1 and terbinafine-sensitive mutant KLN1(pL37P). Strains were grown at 30°C in YPD medium to early log phase. After the inhibition of de novo protein biosynthesis by the addition of cycloheximide, samples of 8 OD600 equivalents were removed at the indicated time points (t). Whole-cell extracts were prepared by NaOH disruption, followed by TCA precipitation. Aliquots were subjected to Western blot analysis with anti-Erg1p antibodies and anti-Sui2p antibodies as the loading control.

  • FIG. 5.
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    FIG. 5.

    Sterol compositions of S. cerevisiae KLN1 strains carrying the wild-type ERG1 gene (NS1) and various erg1 alleles. Cultures were grown for 8 h in the presence of [14C]acetate in YPD medium to log phase. (A) Cellular lipids were isolated and separated by TLC as described in Materials and Methods. Relative radioactivity was expressed as the percentage of the label in neutral lipids. (B) Analysis of nonsaponifiable lipids by two-dimensional TLC. Radioactivity was determined by liquid scintillation counting of the spots corresponding to ergosterol, lanosterol, and squalene.

  • FIG. 6.
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    FIG. 6.

    Cross sensitivity of S. cerevisiae KLN1 strains carrying various erg1 alleles. (A to C) In the presence of the indicated inhibitors, growth in liquid cultures was monitored after 24 h at 30°C by measuring the OD600; (D) overnight cultures of the strains were prepared at 30°C in YPD medium. The OD600 of the overnight cultures was adjusted to 0.1 (100), and 5 μl of each of the 100 to 10−3 dilutions was spotted on YPD agar plates, with or without indicated inhibitor, and grown for 2 days at 30°C.

  • FIG. 7.
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    FIG. 7.

    Homology model of Erg1p from S. cerevisiae, based on the crystal structure of PHBH. (A) Schematic representation of the three-dimensional model. The FAD cofactor is shown as a yellow sticks model. C-terminal residues 440 to 496, which are predicted to form two α helices but which have no corresponding part in PHBH, are not shown. (B) Close-up view of the core cleft of Erg1p with the FAD cofactor. Amino acid side chains, for which mutations are described in the text, are displayed as CPK models (C, orange; N, blue; O, red). (C) Close-up view of a cluster of residues, alterations of which lead to terbinafine-resistant variants of Erg1p. This figure was prepared by using PyMol (http://www.pymol.org ).

Tables

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  • TABLE 1.

    Strains and plasmids used in this study

    Strain or plasmidGenotype or descriptionSource or reference
    E. coli XL1 endA1 hsdR17 (hsdR hsdM+) supE44 thi-1 recA1 gyrA96 relA1 D(lac) [F′ proAB lacIqZDM15 Tn10 (Tetr)]Stratagene
    S. cerevisiae KLN1 MAT a ERG1::URA3 leu2 ura3 trp1 14
    Plasmids
        pRS315Centromere yeast/E. coli shuttle vectorK. Kuchler
        pBIG12.3-kb PstI fragment with the wild-type ERG1 gene in pBluescript 15
        pNS12.3-kb PstI fragment with the wild-type ERG1 gene in pRS315 15
        pG25SpRS315 with 2.3-kb PstI fragment with allelic erg1G25S gene generated by site-directed mutagenesisThis study
        pG27SpRS315 with 2.3-kb PstI fragment with allelic erg1G27S gene generated by site-directed mutagenesisThis study
        pG30SpRS315 with 2.3-kb PstI fragment with allelic erg1G30S gene generated by random mutagenic PCRThis study
        pL37PpRS315 with 2.3-kb PstI fragment with allelic erg1L37P gene generated by random mutagenic PCRThis study
        pR269GpRS315 with 2.3-kb PstI fragment with allelic erg1R269G gene generated by random mutagenic PCRThis study
        pD209ApRS315 with 2.3-kb PstI fragment with allelic erg1D209A gene generated by site-directed mutagenesisThis study
        pG210ApRS315 with 2.3-kb PstI fragment with allelic erg1G210A gene generated by site-directed mutagenesisThis study
        pG334ApRS315 with 2.3-kb PstI fragment with allelic erg1G334A gene generated by site-directed mutagenesisThis study
        pD335ApRS315 with 2.3-kb PstI fragment with allelic erg1D335A gene generated by site-directed mutagenesisThis study
        pD335WpRS315 with 2.3-kb PstI fragment with allelic erg1D335W gene generated by site-directed mutagenesisThis study
        pD335FpRS315 with 2.3-kb PstI fragment with allelic erg1D335F gene generated by site-directed mutagenesisThis study
        pD335PpRS315 with 2.3-kb PstI fragment with allelic erg1D335P gene generated by site-directed mutagenesisThis study
  • TABLE 2.

    Sterol intermediate composition in various ERG1 strainsa

    SIb% of total radioactivity incorporated into sterol intermediates:
    pNS1pG27SpG30SpL37PpR269GpG25S
    S55.0 ± 9.073.1 ± 8.082.6 ± 9.093.4 ± 2.069.0 ± 8.599.4 ± 0.5
    SE6.7 ± 2.06.2 ± 2.03.0 ± 2.01.3 ± 0.75.8 ± 1.5NDc
    L30.1 ± 9.016.1 ± 4.04.6 ± 2.01.1 ± 0.218.0 ± 5.5ND
    • ↵ a KLN1 strains carrying the indicated plasmids.

    • ↵ b SI, sterol intermediate; S, squalene; SE, squalene epoxide; L, lanosterol.

    • ↵ c ND, not detected.

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Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine-Sensitive Variants
Christoph Ruckenstuhl, Silvia Lang, Andrea Poschenel, Armin Eidenberger, Pravas Kumar Baral, Peter Kohút, Ivan Hapala, Karl Gruber, Friederike Turnowsky
Antimicrobial Agents and Chemotherapy Dec 2006, 51 (1) 275-284; DOI: 10.1128/AAC.00988-06

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Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine-Sensitive Variants
Christoph Ruckenstuhl, Silvia Lang, Andrea Poschenel, Armin Eidenberger, Pravas Kumar Baral, Peter Kohút, Ivan Hapala, Karl Gruber, Friederike Turnowsky
Antimicrobial Agents and Chemotherapy Dec 2006, 51 (1) 275-284; DOI: 10.1128/AAC.00988-06
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KEYWORDS

Naphthalenes
Saccharomyces cerevisiae
Squalene Monooxygenase

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