title: Characterization of a mutation that results in independence of oxidosqualene cyclase (Erg7) activity from the downstream 3-ketoreductase (Erg27) in the yeast ergosterol biosynthetic pathway.
abstract: In yeast, deletion of ERG27, which encodes the sterol biosynthetic enzyme, 3-keto-reductase, results in a concomitant loss of the upstream enzyme, Erg7p, an oxidosqualene cyclase (OSC). However, this phenomenon occurs only in fungi, as mammalian Erg27p orthologues are unable to rescue yeast Erg7p activity. In this study, an erg27 mutant containing the mouse ERG27 orthologue was isolated that was capable of growing without sterol supplementation (FGerg27). GC/MS analysis of this strain showed an accumulation of squalene epoxides, 3-ketosterones, and ergosterol. This strain which was crossed to a wildtype and daughter segregants showed an accumulation of squalene epoxides as well as ergosterol indicating that the mutation entailed a leaky block at ERG7. Upon sequencing the yeast ERG7 gene an A598S alteration was found in a conserved alpha helical region. We theorize that this mutation stabilizes Erg7p in a conformation that mimics Erg27p binding. This mutation, while decreasing OSC activity still retains sufficient residual OSC activity such that the strain in the presence of the mammalian 3-keto reductase enzyme functions and no longer requires the yeast Erg27p. Because sterol biosynthesis occurs in the ER, a fusion protein was synthesized combining Erg7p and Erg28p, a resident ER protein and scaffold of the C-4 demethyation complex. Both FGerg27 and erg27 strains containing this fusion plasmid and the mouse ERG27 orthologue showed restoration of ergosterol biosynthesis with minimal accumulation of squalene epoxides. These results indicate retention of Erg7p in the ER increases its activity and suggest a novel method of regulation of ergosterol biosynthesis.
authors.affiliation: Biology Department, Indiana University-Purdue University, Indianapolis, 723 West Michigan Street, Indianapolis, IN 46202, USA.
authors.forename: Jacob V
authors.initials: JV
authors.lastname: Layer
authors.forename: Brett M
authors.initials: BM
authors.lastname: Barnes
authors.forename: Yuji
authors.initials: Y
authors.lastname: Yamasaki
authors.forename: Robert
authors.initials: R
authors.lastname: Barbuch
authors.forename: Liangtao
authors.initials: L
authors.lastname: Li
authors.forename: Silvia
authors.initials: S
authors.lastname: Taramino
authors.forename: Gianni
authors.initials: G
authors.lastname: Balliano
authors.forename: Martin
authors.initials: M
authors.lastname: Bard
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004875:Ergosterol / Q000096:biosynthesis* / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D019751:Intramolecular Transferases; C021924:lanosterol synthase; D004875:Ergosterol