Enzymatic activity and ergosterol-mediated transcriptional regulation of yeast squalene epoxidase

Research project P 14415 Characterization of squalene epoxidase in yeast Friederike TURNOWSKY 08.05.2000 Sterols are essential and characteristic components of eukaryotic membranes. The predominant sterol in fungi is ergosterol which is structurally and functionally related to the mammalian sterol, cholesterol. The biosynthesis of ergosterol is well characterized in Saccharomyces cerevisiae, however the regulation of synthesis is not well understood. One essential enzyme in the pathway is squalene epoxidase which is encoded by the ERG1 gene. The enzyme is the target of the highly active antifungal compounds, the allylamines, of which terbinafine is one of the most active derivates. As single point mutation in the structural gene leads to terbinafine resistance. Squalene epoxidase in yeast is localized in the endoplasmatic reticulum (ER) and the so-called lipid particles. However, the protein is not enzymatically active in isolated lipid particles. Addition of ER protein can restore the enzymatic activity, suggesting that this compartment contains a factor, possibly a protein, which is essential for enzymatic activity. When S. cerevisiae is treated with terbinafine cells accumulate squalene and become deprived of ergosterol. Under such conditions the expression of squalene epoxidase is transcriptionally induced. Incubation with other sterol synthesis inhibitors which block the synthesis pathway at later steps also leads to induced ERGT1 expression, suggesting that ergosterol depletion is responsible for regulated gene expression. Deletion analysis of the ERG1 promoter region allowed us to identify a novel DNA sequence element which is involved in ergosterol mediated ERG1 expression. Two lines of investigations are proposed which are directed towards the elucidation of squalene epoxidase activity, the identification of potential accessory proteins and the interaction with the inhibitor terbinafine (aim 1), and the characterization of the transcription factor involved (aim 2). We propose to purify the Erg1 protein and identify interacting proteins by mass spectrometry. The characterization of such proteins should enable us to elucidate the enzymatic epoxidation reaction. The determination of the crystal structure of Erg1p in the presence of terbinafine is also proposed. To identify the transcription factor (aim 2) we propose to perform DANN element. Alternatively we will apply a one-hybrid system to identify DANN binding proteins. Site specifix mutagenesis of the binding site will allow us to elucidate the sequence requirements for transcription factor binding and regulation.