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J. Biol. Chem., Vol. 282, Issue 49, 36010-36023, December 7, 2007

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Old Yellow Enzymes, Highly Homologous FMN Oxidoreductases with Modulating Roles in Oxidative Stress and Programmed Cell Death in Yeast^*

Osama Odat¹, Samer Matta¹, Hadi Khalil¹, Sotirios C. Kampranis, Raymond Pfau, Philip N. Tsichlis, and Antonios M. Makris²

From the Department of Natural Products, Mediterranean Agronomic Institute of Chania, Chania 73100, Greece and the Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, Massachusetts 02111

In a genetic screen to identify modifiers of Bax-dependent lethality in yeast, the C terminus of OYE2 was isolated based on its capacity to restore sensitivity to a Bax-resistant yeast mutant strain. Overexpression of full-length OYE2 suppresses Bax lethality in yeast, lowers endogenous reactive oxygen species (ROS), increases resistance to H₂O₂-induced programmed cell death (PCD), and significantly lowers ROS levels generated by organic prooxidants. Reciprocally, oye2 yeast strains are sensitive to prooxidant-induced PCD. Overexpression and knock-out analysis indicate these OYE2 antioxidant activities are opposed by OYE3, a highly homologous heterodimerizing protein, which functions as a prooxidant promoting H₂O₂-induced PCD in wild type yeast. To exert its effect OYE3 requires the presence of OYE2. Deletion of the 12 C-terminal amino acids and catalytic inactivation of OYE2 by a Y197F mutation enhance significantly survival upon H₂O₂-induced PCD in wild type cells, but accelerate PCD in oye3 cells, implicating the oye2p-oye3p heterodimer for promoting cell death upon oxidative stress. Unexpectedly, a strain with a double knock-out of these genes (oye2 oye3) is highly resistant to H₂O₂-induced PCD, exhibits increased respiratory capacity, and undergoes less cell death during the adaptive response in chronological aging. Simultaneous deletion of OYE2 and other antioxidant genes hyperinduces endogenous levels of ROS, promoting H₂O₂-induced cell death: in oye2 glr1 yeast high levels of oxidized glutathione elicited gross morphological aberrations involving the actin cytoskeleton and defects in organelle partitioning. Altering the ratio of reduced to oxidized glutathione by exogenous addition of GSH fully reversed these alterations. Based on this work, OYE proteins are firmly placed in the signaling network connecting ROS generation, PCD modulation, and cytoskeletal dynamics in yeast.

Received for publication, May 16, 2007 , and in revised form, September 26, 2007.

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ These authors contributed equally to this research and were partially supported by International Centre for Advanced Mediterranean Agronomic Studies/Mediterranean Agronomic Institute of Chania student scholarships.

² To whom correspondence should be addressed: Alsyllion Agrokepiou, Chania 73100, Greece. Tel.: 30-28210-35050; Fax: 30-28210-35001; E-mail: antoniosmakris{at}yahoo.gr.

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