Genetic analyses involving interactions between the ergosterol biosynthetic enzymes, lanosterol synthase (Erg7p) and 3-ketoreductase (Erg27p), in the yeast Saccharomyces cerevisiae

Abstract

SummaryProtein-protein interaction studies in the S. cerevisiae ergosterol biosynthetic pathway suggest that enzymes in this pathway may act as an integrated multienzyme complex. The yeast sterol 3-ketoreductase (Erg27p) required for C-4 demethylation of sterols has previously been shown to also be required for the function of the upstream oxidosqualene cyclase/lanosterol synthase (Erg7p); thus, erg27 mutants accumulate oxidosqualenes as precursors rather than 3-ketosterones. In the present study, we have created various mutations in the ERG27 gene. These mutations include 5 C-terminal truncations, 6 internal deletions, and 32 point mutants of which 14 were obtained by site directed mutagenesis and 18 by random mutagenesis. We have characterized these ERG27 mutations by determining the following: Erg27 and Erg7 enzyme activities, presence of Erg27p as determined by western immunoblots, ability to grow on various sterol substrates and GC sterol profiles. Mutations of the predicted catalytic residues, Y202F and K206A, resulted in the endogenous accumulation of 3-ketosterones rather than oxidosqualenes suggesting retention of Erg7 enzyme activity. This novel phenotype demonstrated that the catalytic function of Erg27p can be separated from its Erg7p chaperone ability. Other erg27 mutations resulted in proteins that were present, as determined by western immunoblotting, but unable to interact with the Erg7 protein. We also classify Erg27p as belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes and demonstrate the possibility of homo -or hetero-dimerization of the protein. This study provides new insights into the role of Erg27p in sterol biosynthesis.

Mapping Condition-Dependent Regulation of Lipid Metabolism inSaccharomyces cerevisiae

“…Erg6p and Erg7p are involved in sterol biosynthesis and are found almost exclusively in lipid particles (Schulz and Prinz 2007) interacting with Are1p and Are2p, respectively. Because Are1p and Are2p catalyze SE biosynthesis, our data are consistent with the hypothesis that Erg6p and Erg7p may play a regulatory role in SE synthesis (Czabany et al 2007; Teske et al 2008). …”

Interactions of oxidosqualene cyclase (Erg7p) with 3-keto reductase (Erg27p) and other enzymes of sterol biosynthesis in yeast

“…Our previous [9,10] and present results confirm this prediction, suggesting a subtle bidirectional relationship between Erg7p and the C-4 demethylation apparatus. The bidirectional control mechanism between the demethylation complex and oxidosqualene cyclase can be depicted as follows: the complex, formed by Erg25p, Erg26p, Erg27p and Erg28p, controls the functionality of oxidosqualene cyclase through a direct chaperone-like action of Erg27p toward Erg7p, and additionally, oxidosqualene cyclase controls the functionality of the complex by interacting with component(s) other than Erg27p.…”

“…In yeast, interactions involving OSC are less defined although recent studies have demonstrated that OSC is not a solitary protein. The OSC enzyme directly interacts with another enzyme of sterol biosynthesis, the 3-ketosterol reductase (Erg27p), an enzyme involved in C-4 demethylation of intermediates of ergosterol biosynthesis [9,10]. In yeast cells in which the ERG27 gene is deleted, oxidosqualene cyclase (Erg7p) is inactive and thus the “tailoring section” of sterol biosynthesis, which includes the 3-ketoreductase-catalyzed step, appears to affect the step which generates the steroid nucleus.…”

Divergent interactions involving the oxidosqualene cyclase and the steroid-3-ketoreductase in the sterol biosynthetic pathway of mammals and yeasts

“…The wild-type SCY876 strain was grown to early stationary phase at 30°C in YPD medium (1% yeast extract, 2% peptone, 2% glucose). BTY6-5-3 [14] and SDG115 [9] ( erg27 ) strains were supplemented with ergosterol (0.02 mg/mL). STY35, STY36 and STY37 strains were grown in synthetic complete medium without uracil (SC-UraD: 0.17% yeast nitrogen base, 0.2% amino acids, 0.5% ammonium sulphate, 2% glucose).…”

“…Yeast cells lacking the ERG27 gene, show a concomitant loss of the oxidosqualene cyclase (OSC) activity and complete ablation of sterol synthesis. Teske et al [14] previously showed that a catalytically inactive Erg27p retains Erg7p protective function resulting in endogenous accumulation of 3-ketosteroids; however, the basis of the molecular interaction between these two enzymes remains elusive.…”