ACTIVATION OF THE UNCOUPLING PROTEIN BY FATTY-ACIDS IS MODULATED BY MUTATIONS IN THE C-TERMINAL REGION OF THE PROTEIN

The transport properties of the uncoupling protein (UCP) from brown ad ipose tissue have been studied in mutants where Cys304 has been replac ed by either Gly, Ala, Ser, Thr, Ile or Trp. This position is only two residues away from the C-terminus of the protein, a region that faces the cytosolic side of the mitochondrial inner membrane. Mutant protei ns have been expressed in Saccharomyces cerevisiae and their activity determined in situ by comparing yeast growth rates in the presence and absence of 2-bromopalmitate. Their bioenergetic properties have been studied in isolated mitochondria by determining the effects of fatty a cids and nucleotides on the proton permeability and NADH oxidation rat e. It is revealed that substitution of Cys304 by non-charged residues alters the response of UCP to fatty acids. The most effective substitu tion is Cys for Gly since it greatly enhances the sensitivity to palmi tate, decreasing threefold the concentration required for half-maximal stimulation of respiration. The opposite extreme is the substitution by Ala which increases twofold the half-maximal concentration. We conc lude that the C-terminal region participates in the fatty acid regulat ion of UCP activity. The observed correlation between yeast growth rat es in the presence of bromoplamitate and the calculated activation con stants for respiration in isolated mitochondria validates growth analy sis as a method to screen the in situ activity of UCP mutants.