Molecular Cloning Of Amphioxue Uncoupling Protein And The Study About UCP1Structure-function Relationship By Site Directed Mutation

Uncoupling proteins (UCPs) belong to the superfamily of mitochondrial anion-carrier proteins and can dissipate the mitochondrial membrane potential generated during electron transportinto heat. Five distinct isoforms of uncoupling protein, including UCP1to UCP5, have been identified in mammals, UCPlis typically expressed in brown adipose tissue (BAT) of newbornhumans, small mammals and hibernating mammals, and its majorrole is to execute non-shivering thermogenesis by catalyzing protonleak, there is evidence that rat and mouse thymi also containUCP1. UCP2and UCP3exhibit much wider expressionpattern than UCP1, but their physiological role remains controversial, except that they can reduce ROS production under certainstressed conditions, UCP4and UCP5are specifically expressedin brain, but typical UCP1-like biochemical properties of theseproteins have not yet been demonstrated and phylogenetic inferenceexcludes them from the core UCP protein family. Despite that bioinformatics studies have implied the existence of uncouplingproteins in certain invertebrates, the animal uncoupling proteins identified so far are exclusively found in vertebrates, and the occurrence of invertebrate UCPs lacks experimental evidence. The present study describes the molecular cloning of a novel cDNA fragment from amphioxus (Branchiostoma belcheri) encoding a343-amino acid protein that is highly homologous to mamalian uncoupling proteins (UCP), this protein is therefore named amphioxus UCP. To further assess the functional similarity of amphioxus UCP to mammalian UCP1and2, the amphioxus UCP, rat UCP1, and human UCP2were separately expressed in Saccharomyces cerevisiae, and the recombinant yeast mitochondria were isolated and assayed for the state4respiration rate and proton leak. UCP1increased the state4respiration rate by2.8-fold, and the uncoupling activity was strongly inhibited by GDP, while UCP2and amphioxus UCP only increased the state4respiration rate by1.5-fold and1.7-fold in a GDP-insensitive manner, moreover, the proton leak kinetics of amphioxus UCP was very similar to UCP2, but much different from UCP1. In conclusion, the amphioxus UCP has a mild, unregulated uncoupling activity in the yeast system, which resembles mammalian UCP2, but not UCP1.We believed that all the vertebrates UCPs may come from a common ancestor, according to our sturdy on Amphioxus UCP. UCP1, UCP2and UCP3diverged from the common ancestor because of the gene/genome duplication at the origin of vertebrates. We collected73protein and nucleotide sequences from three databases, and did phylogenetic analysis. The phylogenetic tree clearly showed the evolutionary rate of UCP1in the ancestral eutherian was accelerated under the positive Darwinian selection. We tried to find some amino acids that are only special for ancestral entherian UCP1and study the structure-function relationship by site-directed mutation and yeast heterologous expression system. Finally, we found six amino acids in related to the proton leak function of UCP1and this result also proved the accelerated evolution of UCP1.