Abstract
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.
Highlights
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 specifically the 32-kDa potato UCP (StUCP) gene
Immunological Characterization of StUCP and Detection Limits—To characterize UCP in potato tuber mitochondria, we used an antibody raised against mammalian UCP1 that was first tested for its ability to recognize the 32-kDa potato UCP (StUCP) expressed in yeast
To characterize the functional aspects of a plant uncoupling protein (StUCP), the gene encoding this protein was expressed in S. cerevisiae and the level of peptide found in the mitochondria was probed using antimammalian UCP1 antibodies
Summary
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. 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. This fatty acid-induced uncoupling was insensitive to nucleotides. 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 level in yeast mitochondria. Similarities in behavior and properties between StUCP expressed in yeast and PUMP are discussed
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