Sequencing of Rabbit Brown Adipose Tissue Uncoupling Protein cDNA: Characterization of Rat and Rabbit Uncoupling Protein mRNAs

Abstract

A cDNA clone encoding the entire amino acid sequence of rabbit brown adipose tissue uncoupling protein has been isolated and sequenced. The coding region of this cDNA is 80.6% identical to that of the rat uncoupling protein cDNA. In contrast to rat uncoupling protein for which there are two mRNAs of 1500 and 2000 nucleotides there is only one rabbit uncoupling protein mRNA of 2000 nucleotides. Whereas the rat cDNA hybridizes more strongly to the shorter rat uncoupling protein mRNA the rabbit cDNA hybridizes more strongly to the longer rat uncoupling protein mRNA. Primer extension and Northern blot analysis were performed to try to account for the difference of 430 ± 75 nucleotides between the two rat uncoupling protein mRNAs. Northern blot analysis indicated the presence of 355 more nucleotides in the 3'-untranslated region of the 2000 nucleotide long rat uncoupling protein mRNA than in the 1500 nucleotide long rat uncoupling protein mRNA. The two rat uncoupling protein mRNAs could therefore arise by differential processing. Primer extension revealed that the two rat uncoupling protein mRNAs have a 5'-untranslated region of approximately 186 nucleotides. The deduced amino acid sequence of rabbit UCP is 86% identical with both the rat and hamster proteins. Several regions are conserved in all three uncoupling proteins. The two longest regions of conservation are residues 52 to 69 and 82 to 100 of the mature proteins and correspond to two of several basic regions of the protein that have been suggested as possible targeting sequences. These conserved regions fall within amino acids 52 to 104 of the mature rat protein, which has been shown by others to target a passenger protein to mitochondria. Helical wheel diagrams that correspond to residues 52 to 68 and residues 72 to 92 reveal possible amphiphilic α-helical formations that may be involved in targeting. Regions corresponding to those conserved in the three UCPs are also conserved in three mammalian ADP/ATP carriers and may indicate a common role for these regions, perhaps including targeting. There is almost complete conservation of lysine, arginine, and cysteine residues that are thought to be involved in nucleotide binding and proton transport in the three UCPs. There is a threonine to alanine change at the carboxyl-terminus of the rabbit protein compared to the rat protein. This amino acid difference may explain the differential reactivities of rabbit and rat UCP with an antibody preparation against rat UCP.