EXPRESSION OF WILD-TYPE AND MUTANT FORMS OF ARABIDOPSIS UNCOUPLING PROTEIN IN YEAST

Susan K. Cousins, Heidi A. Dombrowski, Midori R. Harris, and Maureen R. Hanson
Section of Genetics and Development, Cornell University, Ithaca, NY

The mitochondrial uncoupling protein, which disassociates the electron transport chain from ATP synthesis, was previously believed to be exclusive to mammalian brown fat and functional under some special conditions such as hibernation and cold tolerance. Recent work has shown that additional UCP homologues are expressed in other parts of the mammalian body [1,2]. Furthermore, UCP activity has been discovered in plants [3,4], indicating that the uncoupling protein may play a more fundamental, evolutionarily conserved role in energy metabolism than previously believed. Like UCP in mammals, plant UCP upregulates transport of protons across the inner mitochondrial membrane in the presence of fatty acids. This effect of fatty acids may be inhibited by dinucleotides. We obtained and sequenced a cDNA clone from the Arabidopsis Biological Resource Center which encodes a plant uncoupling protein with high sequence similarity to animal uncoupling proteins. RNA blot hybridization with the cDNA clone as probe indicates that uncoupling protein is expressed in open flowers and floral buds of various ages in Arabidopsis. To further analyze the regulation of uncoupling protein in plants, we have generated several mutations in the nucleotide binding region and generated transgenic yeast lines expressing these mutant forms of UCP. Flow cytometry of yeast cell populations and spectrofluorimetry of mitochondria purified from these cells were used to determine the effect of the mutations on uncoupling activity.

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[3] Vercesi et al. (1995) Nature 375: 24.
[4] Jezek et al. (1996) J. Biol. Chem. 271: 32743-32748.