Activation and Function of Mitochondrial Uncoupling Protein in Plants

Anna M. O. Smith, R. George Ratcliffe, Lee J. Sweetlove
2004 Journal of Biological Chemistry  
Plant mitochondrial uncoupling protein (UCP) is activated by superoxide suggesting that it may function to minimize mitochondrial reactive oxygen species (ROS) formation. However, the precise mechanism of superoxide activation and the exact function of UCP in plants are not known. We demonstrate that 4-hydroxy-2-nonenal (HNE), a product of lipid peroxidation, and a structurally related compound, trans-retinal, stimulate a proton conductance in potato mitochondria that is inhibitable by GTP (a
more » ... bitable by GTP (a characteristic of UCP). Proof that the effects of HNE and trans-retinal are mediated by UCP is provided by examination of proton conductance in transgenic plants overexpressing UCP. These experiments demonstrate that the mechanism of activation of UCP is conserved between animals and plants and imply a conservation of function. Mitochondria from transgenic plants overexpressing UCP were further studied to provide insight into function. Experimental conditions were designed to mimic a bioenergetic state that might be found in vivo (mitochondria were supplied with pyruvate as well as tricarboxylic cycle acids at in vivo cytosolic concentrations and an exogenous ATP sink was established). Under such conditions, an increase in UCP protein content resulted in a modest but significant decrease in the rate of superoxide production. In addition, 13 C-labeling experiments revealed an increase in the conversion of pyruvate to citrate as a result of increased UCP protein content. These results demonstrate that under simulated in vivo conditions, UCP is active and suggest that UCP may influence not only mitochondrial ROS production but also tricarboxylic acid cycle flux. Mitochondrial uncoupling proteins are integral membrane proteins that reside in the inner mitochondrial membrane and belong to a large family of mitochondrial anion carriers (1). They catalyze a proton conductance that results in the dissipation of the proton gradient across the inner mitochondrial membrane. As such, they partially uncouple electron transport from ATP synthesis. Uncoupling protein 1 (UCP1) 1 was first identified in mammalian brown adipose tissue, where it mediates an uncoupled respiration of fatty acids to generate heat for thermogenesis (2). It makes sense, therefore, that UCP1 is activated by fatty acids, and indeed a proton conductance that is stimulated by fatty acids and inhibited by nucleotides (which act as inhibitors of UCPs) has long been seen as diagnostic of UCP activity (3). A number of homologues of UCP1 exist in animals (2) as well as higher plants (4) , fungi and more primitive organisms (5). The function of these homologues is much debated, but their expression in non-thermogenic tissues means that it is unlikely to be related to heat production. A major advance in our understanding of uncoupling protein function was the discovery that fatty acids do not directly activate UCP. Instead, it was shown that exogenously generated superoxide (in the presence of fatty acids) activates UCPs in both animals (6) and plants (7) . To activate UCP, superoxide must be present in the matrix of the mitochondrion, and thus exogenous superoxide must somehow cross the mitochondrial inner membrane (8). Superoxide is an upstream component of the activation pathway of UCP. The end point of this pathway appears to be products of lipid peroxidation such as 4-hydroxy-2-nonenal (HNE) that contain reactive alkenal groups (9). Such compounds are potent activators of animal UCPs. A mechanism has been proposed in which superoxide in the matrix reacts with free Fe 2ϩ (which is possibly formed as a result of superoxide attack of iron sulfur-containing proteins such as aconitase) to form the highly reactive hydroxyl radical. This radical initiates carbon-centered lipid radical formation leading to lipid peroxidation that ultimately results in aldehyde degradation products such as HNE that activate UCP (10). Given that mild uncoupling of mitochondria dramatically reduces superoxide formation by the electron transport chain (11), this pathway provides an elegant feedback mechanism by which UCP may act to limit the extent of ROS production in mitochondria. Although plant UCPs have been shown to be activated by superoxide (7) , it is not known whether this superoxide activation proceeds via the HNE pathway that occurs in animals. It is important to establish whether HNE activation of UCP is a feature that is conserved during evolution from plants to animals or whether it evolved since the divergence of the two kingdoms. The former would imply a conservation of function between plant and animal UCP, and thus investigations into the role of UCP in plants may have implications for animal UCP1 homologue function. The latter case (i.e. superoxide, but not HNE activation of UCP is conserved between plants and animals) may imply that UCP function in animals is more specifically related to lipid peroxidation rather than ROS per se. A relevant suggestion in this regard is that UCP might function as a transporter of oxidized fatty acid molecules (12) although there is not, as yet, any experimental evidence to support this idea.
doi:10.1074/jbc.m408920200 pmid:15456782 fatcat:x2ierybcyncbrovrx575opvf7i