Hasmukh V. Patel

Identification by Site-directed Mutagenesis of Three Arginines in Uncoupling Protein That Are Essential for Nucleotide Binding and Inhibition*

Primary regulation of uncoupling protein is mediated by purine nucleotides, which bind to the protein and allosterically inhibit fatty acid-induced proton transport. To gain increased understanding of nucleotide regulation, we evaluated the role of basic amino acid residues using site-directed mutagenesis. Mutant and wild-type proteins were expressed in yeast, purified, and reconstituted into liposomes. We studied nucleotide binding as well as inhibition of fatty acid-induced proton transport in wild-type and six mutant uncoupling proteins. None of the mutations interfered with proton transport. Two lysine mutants and a histidine mutant had no effect on nucleotide binding or inhibition. Arg83 and Arg182 mutants completely lost both the ability to bind nucleotides and nucleotide inhibition. Surprisingly, the Arg276 mutant exhibited normal nucleotide binding, but completely lost nucleotide inhibition. To account for this dissociation between binding and inhibition, we propose a three-stage binding-conformational change model of nucleotide regulation of uncoupling protein. We have now identified three nucleotides by site-directed mutagenesis that are essential for nucleotide interaction with uncoupling protein.

Loss of brown adipose tissue uncoupling protein mRNA on deacclimation of cold-exposed rats

The effect of environmental temperature on the level of uncoupling protein mRNA from rat brown adipose tissue was examined using a cDNA probe. A 4.4 fold increase in the mRNA level was observed after 1 day exposure of rats to 6 degrees C, which was followed by a slow loss with longer times of exposure. When rats were returned to a thermoneutral environment, there was a dramatic loss of uncoupling protein mRNA within 1 day. Comparison wih poly(A)+ RNA levels suggest that the response to temperature is specific for uncoupling protein mRNA.

Synthesis in vitro of rat brown adipose tissue 32000 Mr protein

The synthesis of the mitochondrial inner membrane 32 000 M r protein from rat brown adipose tissue was examined. Polysomes from the tissue were translated in a reticulocyte lysate protein‐synthesizing system and newly‐synthesized protein isolated with a monospecific antibody against the 32 000 M r protein. The newly‐synthesized protein had the same relative molecular mass as the mature protein. It was taken up by mitochondria isolated from Chinese hamster ovary cells into a form resistant to trypsin.

A putative precursor of rat liver mitochondrial malate dehydrogenase.

Mitochondria have their own genetic system but the majority of mitochondrial proteins are coded by nuclear DNA, synthesized on cytosolic ribosomes and imported into mitochondria [ 1,2]. Little is known of the mechanism of import [3-61. There is some evidence that the mature form or subunits of the mitochondrial matrix proteins aspartate aminotransferase [6-91 and malate dehydrogenase (EC 1 .l .1.37) [6,1 O-l 21 can be taken up into the matrix of isolated mitochondria. However, a number of mitochondrial matrix proteins including aspartate aminotransferase [ 131 are synthesized in precursor form [3,5]. Here, we report a putative precursor or rat liver mitochondrial malate dehydrogenase which is synthesized in a reticulocyte lysate primed with either free polysomes or total RNA from rat liver.

Immunological detection of cDNA clones encoding the uncoupling protein of brown adipose tissue: Evidence for an antigenic determinant within the C-terminal eleven amino acids

Poly(A)+ RNA was isolated from brown adipose tissue of cold acclimated rats and a fraction enriched for uncoupling protein mRNA was used to generate a cDNA library in pBR 322, Immunological screening of 1,500 colonies with an affinity-purified antiserum against the uncoupling protein yielded five positive clones, pUCPratl–5. Clone pUCPrat2 encoded the C-terminal 54 amino acids of rat uncoupling protein and exhibited 90% amino acid homology with the hamster protein. Clones pUCPrat3–5 encoded only the C-terminal 11 amino acids suggesting that an antigenic determinant lies within this sequence.

Effect of a cold stimulus on the synthesis of uncoupling protein in brown adipose tissue of rats and newborn rabbits.

Rats are known to respond to a cold stimulus by increasing the activity and amount of the uncoupling protein in brown adipose tissue. A 48 h cold stimulus was found to increase the synthesis of uncoupling protein 3.g-fold in 4–5 week old rats whereas no change was observed with newborn rabbits. The lack of response in the latter case may reflect a difference between rabbits and rats or that synthesis is already maximal in newborn rabbits.

Cybrid formation with recipient cell lines containing dominant phenotypes.

A clone of Chinese hamster ovary (CHO) cells, BT3,resistant to Tevenel, the sulfamoyl analog of chloramphenicol has been isolated. Resistance was found to be at the mitochondrial level and was shown to be cytoplasmically inherited. This marker was then used to develop a method by which a cell line possessing a dominant nuclear mutation (resistance to 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole, DRB) could be used as a recipient in cybrid formation. The unique feature in this procedure was the removal of nucleated cells from the cytoplasts by passage through unipore filters. The dominant character of the DRB- and Tevenel-resistant phenotypes permitted the selection of cybrids immediately after fusion. This initially increased the frequency of cybrid clones 16-fold as compared to a recipient cell line possessing a recessive marker. The possibility of extending the method to recipient cells lacking a selectable drug-resistance marker is discussed.

MEASUREMENT OF THERMOGENIN SYNTHESIS IN BROWN ADIPOSE TISSUE (BAT)

Thermogenin is a mitochondrial inner membrane protein which mediates non-shivering thermogenesis in BAT. It has a molecular mass of 32,000 Da and acts by uncoupling mitochondria to produce heat. We report a method of measuring its synthesis. Polysomes were isolated from BAT of developing rabbits and translated in a reticulocyte protein synthesizing system. Newly synthesized thermogenin was isolated with monospecific antibodies. This technique has been applied in developing rabbits to demonstrate a 5-fold increase in thermogenin synthesis before birth, and a gradual decrease after birth.The technique provides a method to study other perinatal influences on thermogenin synthesis such as the effect of postnatal environmental temperature. In preliminary experiments thermogenin has been purified from human neonatal BAT.