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Primary regulation of uncoupling protein is mediated by purine nucleotides, which bind to the protein and allosterically inhibit fatty acid-induced proton transport. To gain increased understanding of nucleotide regulation, we evaluated the role of basic amino acid residues using site-directed mutagenesis. Mutant and wild-type proteins were expressed in yeast, purified, and reconstituted into liposomes. We studied nucleotide binding as well as inhibition of fatty acid-induced proton transport in wild-type and six mutant uncoupling proteins. None of the mutations interfered with proton transport. Two lysine mutants and a histidine mutant had no effect on nucleotide binding or inhibition. Arg83 and Arg182 mutants completely lost both the ability to bind nucleotides and nucleotide inhibition. Surprisingly, the Arg276 mutant exhibited normal nucleotide binding, but completely lost nucleotide inhibition. To account for this dissociation between binding and inhibition, we propose a three-stage binding-conformational change model of nucleotide regulation of uncoupling protein. We have now identified three nucleotides by site-directed mutagenesis that are essential for nucleotide interaction with uncoupling protein.

The uncoupling protein is one of a family of mitochondrial transport proteins involved in energy metabolism. It dissipates oxidative energy to generate heat, either by catalyzing proton transport directly or by catalyzing fatty acid anion transport, thus enabling fatty acids to act as cycling protonophores. This transport process is tightly regulated by purine nucleotides. We have expressed uncoupling protein in yeast and examined its proton transport activity after its reconstitution into proteoliposomes. A directed change of Arg276 to Leu or Gln completely abolished nucleotide inhibition of protonophoretic action of the reconstituted mutant uncoupling proteins without affecting the transport process. Arg276 is the first residue of functional importance to be identified in uncoupling protein. Mutation of the homologous residue in the yeast ADP/ATP translocator prevented the growth of yeast on a nonfermentable carbon source, presumably by interfering with nucleotide exchange (Nelson, D. R., Lawson, J. E., Klingenberg, M., and Douglas, M. G. (1993) J. Mol. Biol. 230, 1159-1170). Demonstration of the essential role of a single homologous residue in protein-nucleotide interaction within these two transporters is the first direct evidence that uncoupling protein and the ADP/ATP translocator belong to the same gene family.

Small mammals, including human infants, rely on nonshivering thermogenesis for a substantial portion of their body heat during exposure to cold. This thermogenesis is mediated in large part by the uncoupling protein, which is found exclusively within the inner membrane of brown adipose tissue mitochondria. The sole function of uncoupling protein is to provide a regulated transport pathway for electrophoretic back-flux of H+ ions into the mitochondrial matrix, thereby dissipating the protonmotive force and producing heat. Thus, uncoupling protein is unique with respect to both its physiological role and its tissue expression. We have now achieved high level expression of rat uncoupling protein in yeast, with import into yeast mitochondria at levels, 70-100 micrograms/mg of mitochondrial protein, similar to those observed in brown adipose tissue mitochondria from cold-adapted rats. When the expressed protein was purified and reconstituted into liposomes, the proteoliposomes exhibited GDP-sensitive proton and chloride uniports that were inhibited by GDP with Ki values similar to those obtained with native protein. Moreover, the molecular activities of the expressed protein with respect to Cl- and H+ transport were indistinguishable from those of native protein. The availability of unlimited amounts of functional, expressed uncoupling protein will now permit application of site-directed mutagenesis to the many intriguing aspects of uncoupling protein structure and function.