IDENTIFICATION BY SITE-DIRECTED MUTAGENESIS OF 3 ARGININES IN UNCOUPLING PROTEIN THAT ARE ESSENTIAL FOR NUCLEOTIDE-BINDING AND INHIBITION

Primary regulation of uncoupling protein is mediated by purine nucleot ides, which bind to the protein and allosterically inhibit fatty acid- induced proton transport. To gain increased understanding of nucleotid e regulation, we evaluated the role of basic amino acid residues using site-directed mutagenesis. Mutant and wildtype proteins were expresse d in yeast, purified, and reconstituted into liposomes. We studied nuc leotide binding as well as inhibition of fatty acid-induced proton tra nsport in wild-type and six mutant uncoupling proteins. None of the mu tations interfered with proton transport. Two lysine mutants and a his tidine mutant had no effect on nucleotide binding or inhibition, Arg(8 3) and Arg(182) mutants completely lost both the ability to bind nucle otides and nucleotide inhibition. Surprisingly, the Arg(276) mutant ex hibited normal nucleotide binding, but completely lost nucleotide inhi bition. To account for this dissociation between binding and inhibitio n, Foe propose a three-stage binding-conformational change model of nu cleotide regulation of uncoupling protein. We have now identified thre e nucleotides by site-directed mutagenesis that are essential for nucl eotide interaction with uncoupling protein.