Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli

Borecký, Jiřı́; Maia, Ivan de Godoy; Costa, Alexandre Dias Tavares; Ježek, Petr; Chaimovich, Hernán; Andrade, Paula Bresciani Martins de; Vercesi, Anı́bal E.; Arruda, Paulo

doi:10.1016/s0014-5793(01)02835-6

Cited by 54 publications

(36 citation statements)

References 31 publications

(79 reference statements)

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“…We showed that StUCP yeast mitochondria exhibited a diminished resting potential and a high sensitivity to uncoupling by LA. The StUCP-dependent uncoupling activity measured in yeast (expressed as LA-stimulated oxidation rate) was similar in mitochondria from yeast expressing StUCP (this work) and mammalian UCP2 (16) (7,29). StUCP protonophoric activity in yeast mitochondria is also in agreement with the ability of plant UCP reconstituted in proteoliposomes to collapse the proton gradient, as evidenced with AtUCP1 expressed in E. coli (7) or with the PUMP fraction isolated from potato mitochondria (1,30).…”

Section: Discussionsupporting

confidence: 76%

“…In addition, various cDNAs coding for putative UCPs were identified in plants such as potato, Arabidopsis thaliana, and skunk cabbage (Symplocarpus foetidus), a thermogenic plant from the Aracea family (3)(4)(5)(6). Recently, the Arabidopsis thaliana AtUCP1 gene was expressed in Escherichia coli and the isolated protein reconstituted into liposomes (7). Like mammalian UCPs, the plant UCP 1 requires free fatty acids for its activity and is inhibited by purine nucleotides (GDP, ATP, GTP, or ADP) (7)(8)(9)(10).…”

mentioning

confidence: 99%

“…Recently, the Arabidopsis thaliana AtUCP1 gene was expressed in Escherichia coli and the isolated protein reconstituted into liposomes (7). Like mammalian UCPs, the plant UCP 1 requires free fatty acids for its activity and is inhibited by purine nucleotides (GDP, ATP, GTP, or ADP) (7)(8)(9)(10). At the mitochondrial level, it has been proposed that ATP, GTP, or GDP were absolutely required to get a fully coupled state in potato tuber mitochondria oxidizing succinate (1,8).…”

mentioning

confidence: 99%

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Alteration of Plant Mitochondrial Proton Conductance by Free Fatty Acids

Cabassa¹,

Mesneau²,

Miroux³

et al. 2002

Journal of Biological Chemistry

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We characterized the uncoupling activity of the plant uncoupling protein from Solanum tuberosum (StUCP) using mitochondria from intact potato tubers or from yeast (Saccharomyces cerevisiae) expressing the StUCP gene. Compared with mitochondria from transfected yeast, StUCP is present at very low levels in intact potato mitochondrial membranes (at least thirty times lower) as shown by immunodetection with anti-UCP1 antibodies. Under conditions that ruled out undesirable effects of nucleotides and free fatty acids on uncoupling activity measurement in plant mitochondria, the linoleic acid-induced depolarization in potato mitochondria was insensitive to the nucleotides ATP, GTP, or GDP. In addition, sensitivity to linoleic acid was similar in potato and in control yeast mitochondria, suggesting that uncoupling occurring in potato mitochondria was because of a UCP-independent proton diffusion process. By contrast, yeast mitochondria expressing StUCP exhibited a higher sensitivity to free fatty acids than those from the control yeast and especially a marked proton conductance in the presence of low amounts of linoleic acid. However, this fatty acid-induced uncoupling was also insensitive to nucleotides. Altogether, these results suggest that uncoupling of oxidative phosphorylation and heat production cannot be the dominant feature of StUCP expressed in native potato tissues. However, it could play a role in preventing reactive oxygen species production as proposed for mammalian UCP2 and UCP3.The discovery in plants of a mitochondrial uncoupling protein resembling uncoupling proteins from mammals raised the possibility of a new uncoupling mechanism involving a transport pathway for backflow of protons into the matrix of plant mitochondria (1, 2). Indeed, a protein fraction containing a purine nucleotide-sensitive, free fatty acid-activated uncoupling activity, called PUMP, has been isolated from potato tuber mitochondria and characterized after reconstitution in proteoliposomes (1). In addition, various cDNAs coding for putative UCPs were identified in plants such as potato, Arabidopsis thaliana, and skunk cabbage (Symplocarpus foetidus), a thermogenic plant from the Aracea family (3-6). Recently, the Arabidopsis thaliana AtUCP1 gene was expressed in Escherichia coli and the isolated protein reconstituted into liposomes (7). Like mammalian UCPs, the plant UCP 1 requires free fatty acids for its activity and is inhibited by purine nucleotides (GDP, ATP, GTP, or ADP) (7-10). At the mitochondrial level, it has been proposed that ATP, GTP, or GDP were absolutely required to get a fully coupled state in potato tuber mitochondria oxidizing succinate (1, 8). Similarly, it has been suggested that the fatty acid-induced uncoupling in wheat mitochondria was sensitive to purine nucleotides (11).In this paper, we sought to characterize better the properties of plant UCP-like protein by comparing the fatty acid-dependent uncoupling capacity of either native UCP present in intact potato mitochondria or StUCP expressed at a high lev...

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Section: Discussionsupporting

confidence: 76%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Alteration of Plant Mitochondrial Proton Conductance by Free Fatty Acids

Cabassa¹,

Mesneau²,

Miroux³

et al. 2002

Journal of Biological Chemistry

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“…It is now apparent that UCPs are encoded by small gene families, although consensus on the precise composition of these families has not yet been reached (9,10). Plant UCPs appear to be regulated in a similar fashion to their mammalian counterparts; they catalyze a fatty aciddependent, nucleotide-inhibitable proton conductance (11)(12)(13) and are activated by superoxide and aldehyde products of lipid peroxidation (14,15). Given that formation of superoxide and related reactive oxygen species (ROS) by the respiratory chain is increased nonlinearly at high mitochondrial membrane potential, activation of UCP by superoxide represents a feedback loop by which superoxide production can be regulated.…”

mentioning

confidence: 99%

Mitochondrial uncoupling protein is required for efficient photosynthesis

Sweetlove

Lytovchenko

Morgan

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

227

203

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Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane and dissipate the proton gradient across this membrane that is normally used for ATP synthesis. Although the catalytic function and regulation of plant UCPs have been described, the physiological purpose of UCP in plants has not been established. Here, biochemical and physiological analyses of an insertional knockout of one of the Arabidopsis UCP genes (AtUCP1) are presented that resolve this issue. Absence of UCP1 results in localized oxidative stress but does not impair the ability of the plant to withstand a wide range of abiotic stresses. However, absence of UCP1 results in a photosynthetic phenotype. Specifically there is a restriction in photorespiration with a decrease in the rate of oxidation of photorespiratory glycine in the mitochondrion. This change leads to an associated reduced photosynthetic carbon assimilation rate. Collectively, these results suggest that the main physiological role of UCP1 in Arabidopsis leaves is related to maintaining the redox poise of the mitochondrial electron transport chain to facilitate photosynthetic metabolism. mitochondria ͉ Arabidopsis ͉ electron transport ͉ reactive oxygen species ͉ photorespiration

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“…UCP-like activity has been observed in isolated mitochondria from a number of plant species and the potato UCP activity has been purified and its proton transport properties recovered by reconsitution into liposomes (18). Furthermore, reconstitution of the AtUCP1 gene product into liposomes has provided the first link between a plant UCP gene and proton transport activity (19). However, a recent study has shown that when care is taken to avoid artifacts caused by extraneous effects of nucleotides on other electron transport chain components, the fatty acid-stimulated proton leak of potato mitochondria is not inhibited by nucleotides (20).…”

mentioning

confidence: 99%

Superoxide Stimulates a Proton Leak in Potato Mitochondria That Is Related to the Activity of Uncoupling Protein

Considine

Goodman

Echtay

et al. 2003

Journal of Biological Chemistry

129

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The ability of plant mitochondrial uncoupling proteins to catalyze a significant proton conductance in situ is controversial. We have re-examined conditions that lead to uncoupling of mitochondria isolated from the tubers of potato (Solanum tuberosum). Specifically, we have investigated the effect of superoxide. In the absence of superoxide, linoleic acid stimulated a proton leak in mitochondria respiring NADH that was insensitive to GTP. However, when exogenous superoxide was generated by the addition of xanthine and xanthine oxidase, there was an additional linoleic acid-stimulated proton leak that was specifically inhibited by GTP. Under these conditions of assay (NADH as a respiratory substrate, in the presence of linoleic acid and xanthine/ xanthine oxidase) there was a higher rate of proton conductance in mitochondria from transgenic potato tubers overexpressing the StUCP gene than those from wild type. The increased proton leak in the transgenic mitochondria was completely abolished by the addition of GTP. This suggests that superoxide and linoleic acid stimulate a proton leak in potato mitochondria that is related to the activity of uncoupling protein. Furthermore, it demonstrates that changes in the amount of StUCP can alter the rate of proton conductance of potato mitochondria.

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Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli

Cited by 54 publications

References 31 publications

Alteration of Plant Mitochondrial Proton Conductance by Free Fatty Acids

Alteration of Plant Mitochondrial Proton Conductance by Free Fatty Acids

Mitochondrial uncoupling protein is required for efficient photosynthesis

Superoxide Stimulates a Proton Leak in Potato Mitochondria That Is Related to the Activity of Uncoupling Protein

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