Role of intrahelical arginine residues in functional properties of uncoupling protein (UCP1).

The functional role of the four intrahelical arginines in uncoupling protein (UCP1) from brown adipose tissue were studied in mutants where they were replaced by noncharged residues. Wild-type and mutant UCP1 were expressed in Saccharomyces cerevisiae. As measured in isolated UCP1, nucleotide binding was largely lost in mutants of R83, R182, and R276 occurring in three repeated domains and common to mitochondrial carrier family, whereas mutation of the UCP typical R91 shows normal binding capacity but > 20-fold lower binding affinity and a near loss of pH dependency of binding. In reconstituted UCP1, fatty acid dependent H(+) transport is retained in all four mutants, but inhibition by nucleotide changes according to the binding ability of UCP1. Cl(-) transport is inhibited only by mutations of arginines in the first domain (R83 and R91). Also in isolated mitochondria H(+) transport and respiration with all four mutants is similar to wt, and inhibition by GDP is found only in R91T. The three "regular" arginines are suggested to influence the nucleotide binding site indirectly via a charge network and the "extra" R91 directly via an ion bond with the previously characterised pH sensor E190. The mutants were also used to assess intrahelical control of UCP1. In the yeast cells expressing UCP1, the aerobic growth could be reduced by fatty acid addition only with the nucleotide insensitive mutants. This demonstrates an intracellular control of UCP1 by nucleotides and fatty acids.