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 wildtype 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. Arg
A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein.
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