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IEX-1 (Immediate Early response gene X-1) is a stress-inducible gene. It suppresses production of reactive oxygen species (ROS) and protects cells from apoptosis induced by a wide range of stimuli, but the underlying mechanism is not known. This study reveals that IEX-1 targets the mitochondrial F1Fo-ATPase Inhibitor (IF1) for degradation, resulting in acceleration of ATP hydrolysis, concomitant with reduction in ROS production. A prominent role for IF1 degradation in the function of IEX-1 was corroborated by siRNA-mediated gene silencing of IF1 that recapitulated the effects of IEX-1 on ATP hydrolysis and ROS production. Moreover, progressive C-terminal truncation studies demonstrated that IEX-1 interacted with the C terminus of IF1 and the interaction might render IF1 prone to degradation by an as yet unidentified mitochondrial protease. In support of a physiological importance of IEX-1 in the modulation of IF1 expression, gene-targeted deletion of IEX-1 stabilized IF1 and reduced mitochondrial F1Fo-ATPase activity in vivo. The altered activity of the F1Fo enzyme may account for a metabolic switch from oxidative phosphorylation toward glycolysis in IEX-1 deficient cells. Thus, IEX-1 deficient cells were more susceptible to glucose deprivation than wild type counterparts and displayed increased glucose uptake and lactate production in hypoxic conditions. The cells were also relatively refractory to oligomycin-mediated inhibition of ATP production. The studies offer novel insights into the primary role of IEX-1 in regulating a balance between energy provision and ROS production. Mitochondria are the site for the majority of cellular ATP synthesis, the main source for intracellular reactive oxygen species (ROS) production, and the key machinery for regulating cell death. These three biologic events are tied to the respiratory chain in the inner mitochondrial membrane. The electron transport chain consists of complex I, II, III, and IV, as well as an ATP synthase. The ATP synthase is comprised of the membrane-spanning Fo and the soluble F1 sectors, both of which are multiple protein complex.1,2 The F1Fo enzyme catalyzes the synthesis of ATP or oxidative phosphorylation utilizing the energy produced by the transmembrane electrochemical proton gradient along the respiratory chain. A high membrane potential (Dc m ) in the inner mitochondrial membrane as a consequence of cellular stress would slow the proton flux, giving rise to ROS formation. 3 Depending on the amounts of ROS generated, an intermediate level of ROS can amplify oxidant-sensitive signaling to promote cell proliferation, whereas too much of ROS production causes oxidative stress or mitochondrial membrane disruption that can trigger either apoptosis or necrosis. 4 Under conditions of oxygen deprivation, such as what occurs during ischemia or in the presence of an uncoupler of oxidative phosphorylation, the F1Fo-ATP synthase can switch from an ATP synthase to an ATPase, making it hydrolyze ATP produced in the cytosol by glycolysis. 5To preserve ATP, a nat...