title: Characterizing sterol defect suppressors uncovers a novel transcriptional signaling pathway regulating zymosterol biosynthesis.
abstract: erg26-1ts cells harbor defects in the 4alpha-carboxysterol-C3 dehydrogenase activity necessary for conversion of 4,4-dimethylzymosterol to zymosterol. Mutant cells accumulate toxic 4-carboxysterols and are inviable at high temperature. A genetic screen aimed at cloning recessive mutations remediating the temperature sensitive growth defect has resulted in the isolation of four complementation groups, ets1-4 (erg26-1ts temperature sensitive suppressor). We describe the characterization of ets1-1 and ets2-1. Gas chromatography/mass spectrometry analyses demonstrate that erg26-1ts ets1-1 and erg26-1ts ets2-1 cells do not accumulate 4-carboxysterols, rather these cells have increased levels of squalene and squalene epoxide, respectively. ets1-1 and ets2-1 cells accumulate these same sterol intermediates. Chromosomal integration of ERG1 ERG7 at their loci in erg26-1ts ets1-1 and erg26-1ts and ets2-1 mutants, respectively, results in the loss of accumulation of squalene and squalene epoxide, re-accumulation of 4-carboxysterols and cell inviability at high temperature. Enzymatic assays demonstrate that mutants harboring the ets1-1 allele have decreased squalene epoxidase activity, while those containing the ets2-1 allele show weakened oxidosqualene cyclase activity. Thus, ETS1 and ETS2 are allelic to ERG1 and ERG7, respectively. We have mapped mutations within the erg1-1/ets1-1 (G247D) and erg7-1/ets2-1 (D530N, V615E) alleles that suppress the inviability of erg26-1ts at high temperature, and cause accumulation of sterol intermediates and decreased enzymatic activities. Finally using erg1-1 and erg7-1 mutant strains, we demonstrate that the expression of the ERG25/26/27 genes required for zymosterol biosynthesis are coordinately transcriptionally regulated, along with ERG1 and ERG7, in response to blocks in sterol biosynthesis. Transcriptional regulation requires the transcription factors, Upc2p and Ecm22p.
authors.affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USA.
authors.forename: Melody
authors.initials: M
authors.lastname: Germann
authors.forename: Christina
authors.initials: C
authors.lastname: Gallo
authors.forename: Timothy
authors.initials: T
authors.lastname: Donahue
authors.forename: Reza
authors.initials: R
authors.lastname: Shirzadi
authors.forename: Joseph
authors.initials: J
authors.lastname: Stukey
authors.forename: Silvia
authors.initials: S
authors.lastname: Lang
authors.forename: Christoph
authors.initials: C
authors.lastname: Ruckenstuhl
authors.forename: Simonetta
authors.initials: S
authors.lastname: Oliaro-Bosso
authors.forename: Virginia
authors.initials: V
authors.lastname: McDonough
authors.forename: Friederike
authors.initials: F
authors.lastname: Turnowsky
authors.forename: Gianni
authors.initials: G
authors.lastname: Balliano
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Nickels
mesh_terms: D000483:Alleles; D015152:Blotting, Northern; D015153:Blotting, Western; D049109:Cell Proliferation; D002784:Cholesterol / Q000096:biosynthesis* / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D008401:Gas Chromatography-Mass Spectrometry; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D007763:Lac Operon; D008954:Models, Biological; D009154:Mutation; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D015398:Signal Transduction; D013185:Squalene / Q000031:analogs & derivatives / Q000737:chemistry; D013261:Sterols / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: C002821:2,3-oxidosqualene; D012333:RNA, Messenger; D013261:Sterols; D013185:Squalene; D002784:Cholesterol; D001616:beta-Galactosidase; C015582:zymosterol