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

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.

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.

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.

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.

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.