Chimeric proteins between UCP1 and UCP3: The middle third of UCP1 is necessary and sufficient for activation by fatty acids

Uncoupling protein (UCP) 1 and UCP3 are mitochondrial inner membrane protei ns which both mediate proton leak and thus decrease the mitochondrial trans membrane proton gradient. However, UCP1 and UCP3 differ in their biochemica l regulation. UCP1 is activated by free fatty acids and inhibited by purine nucleotides. Using heterologous expression studies in yeast, UCP3 was foun d to lack, both fatty acid activation and purine nucleotide inhibition. To assess which domains are responsible for the regulation of UCP1 by free fat ty acids and by purine nucleotides and the absence of such regulation in UC P3, chimeric proteins were generated. Given that uncoupling proteins, like all members of the mitochondrial carrier family, possess a tripartite struc ture and consist of three repeated domains of approximately 100 residues, s waps in the three repeated domains were made between UCP1 and UCP3. Regulat ion of the resulting six different chimeric proteins by free fatty acids an d purine nucleotides was studied after heterologous expression in yeast mit ochondria. In this study, it is shown that activation of UCP1 by free fatty acids is mediated by the second repeated domain, since substitution of the second repeat of UCP1 by the equivalent repeat of UCP3 abolishes fatty aci d activation. In contrast, replacing the second repeat of UCP3 by the corre sponding repeated domain of UCP1 results in fatty acid activation similar t o wild type UCP1. The lack, of free fatty acid activation of UCP3 is not du e to the absence of the histidine pair H145 and H147 found in the second re peated domain of UCP1. Furthermore, the findings with respect to purine nuc leotide inhibition are consistent with a significant role of the C-terminal repeated domain of UCP1 in mediating purine nucleotide inhibition. (C) 200 0 Academic Press.