ABSTRACT
A cDNA containing the complete coding sequence of rabbit brown adipose tissue uncoupling protein was isolated and sequenced. The coding region is 80.6% identical to rat UCP cDNA and the protein is about 86% identical to the rat and hamster proteins. Despite the presence of 2 AATAAA polyadenylation consensus sequences in rabbit UCP cDNA, only one rabbit UCP mRNA was detected indicating that only the 3'-downstream signal is used in contrast to rat and mouse where both are used.
Subject(s)
Adipose Tissue, Brown/metabolism , Carrier Proteins , Membrane Proteins/genetics , Protein Processing, Post-Translational , RNA, Messenger/metabolism , Uncoupling Agents/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Ion Channels , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins , Molecular Sequence Data , Poly A/genetics , Poly A/metabolism , Rabbits , Rats , Uncoupling Agents/isolation & purification , Uncoupling Agents/metabolism , Uncoupling Protein 1ABSTRACT
Guanosine diphosphate binding to the uncoupling protein of isolated mitochondria of brown adipose tissue in newborn rabbits was measured as an index of thermogenic activity. The binding was 0.281 +/- 0.022 nmol GDP/mg mitochondrial protein at 1 day of age, 0.214 +/- 0.017 at 3 days, 0.428 +/- 0.038 at 5 days, and 0.208 +/- 0.016 at 7 days. The increase in binding between 3 and 7 days of age suggests that the brown fat has an increased thermogenic capacity at that age. In addition, the potential for synthesis of the uncoupling protein was investigated in 1- to 5-day-old newborn rabbits by probing the total cellular ribonucleic acid for the messenger that codes for uncoupling protein. The amount of uncoupling protein messenger was highest at 1 day of age and declined at least until 5 days of age. Because the amount of uncoupling protein messenger decreased as the GDP binding increased, the results suggest that either the initially translated uncoupling protein was unmasked at about 5 days of age or there was a delay in the incorporation of uncoupling protein into the mitochondrial inner membrane, or both.
Subject(s)
Adipose Tissue, Brown/metabolism , Carrier Proteins , Guanine Nucleotides/metabolism , Guanosine Diphosphate/metabolism , Membrane Proteins/metabolism , Mitochondria/metabolism , RNA, Messenger/analysis , Uncoupling Agents/metabolism , Adipose Tissue, Brown/analysis , Animals , Animals, Newborn , Blotting, Northern , Body Temperature Regulation , Female , Ion Channels , Male , Membrane Proteins/genetics , Mitochondria/analysis , Mitochondrial Proteins , RNA, Messenger/genetics , Rabbits , Uncoupling Agents/genetics , Uncoupling Protein 1ABSTRACT
Brown adipose tissue uncoupling protein (UCP), an integral polytopic protein of the mitochondrial inner membrane, is composed of at least six transmembrane segments whose net hydrophobic character derives from paired amphiphilic helices. The protein is synthesized in the cytoplasm as a polypeptide (307 amino acids) lacking a cleavable targeting (signal) peptide. Deletion mutagenesis and fusion protein constructions revealed the existence of at least two import signals: one lying between UCP precursor amino acids 13-105 and the other downstream of position 101. The former resulted in both targeting and membrane insertion of a fusion protein, whereas the latter targeted UCP 102-307 into the organelle but failed to result in membrane insertion. When a strong matrix-targeting signal derived from precarbamoyl phosphate synthetase was fused to UCP amino acids 169-307 or 52-307 (containing three and five transmembrane domains, respectively), the fusion proteins were efficiently imported to the soluble matrix compartment where correct signal cleavage took place. We suggest that assembly of UCP into the inner membrane follows a coordinate insertion pathway for integration and may use more than one signal sequence to achieve this. In this respect, it might share certain mechanistic features with the insertion of polytopic proteins into the endoplasmic reticulum. The data also suggest, however, that integration of the amino-terminal third of UCP into the inner membrane may be required to help or enhance insertion of the remaining UCP transmembrane domains.
