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J. Biol. Chem., Vol. 280, Issue 43, 35904-35913, October 28, 2005

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Characterizing Sterol Defect Suppressors Uncovers a Novel Transcriptional Signaling Pathway Regulating Zymosterol Biosynthesis^*

Melody Germann1, Christina Gallo1, Timothy Donahue, Reza Shirzadi, Joseph Stukey, Silvia Lang¶, Christoph Ruckenstuhl¶, Simonetta Oliaro-Bosso||, Virginia McDonough, Friederike Turnowsky¶, Gianni Balliano||, and Joseph T. Nickels, Jr.2

From the Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, the Department of Biology, Hope College, Holland, Michigan 49422, the ^¶Institute of Molecular Biology, Biochemistry and Microbiology, Karl-Franzens-University Graz, A-8010 Graz, Austria, and the ^||Dipartimento di Scienza e Tecnologia del Farmaco, Facoltà di Farmacia, Università degli Studi di Torino, Corso Raffaello 31, I-10125, Turin, Italy

erg26-1^ts cells harbor defects in the 4-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 (erg26-1^ts temperature sensitive suppressor). We describe the characterization of ets1-1 and ets2-1. Gas chromatography/mass spectrometry analyses demonstrate that erg26-1^ts ets1-1 and erg26-1^ts 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 erg26-1^ts ets1-1 and erg26-1^ts 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 erg26-1^ts 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.

Received for publication, May 5, 2005 , and in revised form, July 11, 2005.

^* This work was supported by National Institutes of Health Grant HL67401 (to J. T. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102. Tel.: 215-762-1941; Fax: 215-762-4452; E-mail: JN27{at}drexel.edu.

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