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|Title:||Structure/Function Studies of Rat Uncoupling Protein in Yeast Mitochondria|
|Authors:||Murdza-Inglis, Lynn Debra|
|Abstract:||<p>Uncoupling protein (UCP) is one of a family of mitochondrial transport proteins involved in energy metabolism. It is specific to brown adipose tissue mitochondria where it has the primary function in mammals of non-shivering thermogenesis. Heat production is achieved by uncoupling oxidative phosphorylation. It has been suggested that UCP serves the uncoupling role by providing a regulated pathway for proton re-entry back into the mitochondrial matrix. UCP also transports monovalent anions electrophoretically, such as chloride and alkylsulfonates, but the physiological role of anion transport is not yet known, although models based on UCP-catalyzed anion transport for uncoupling have been put forth. Both proton and anion transport are inhibited by purine nucleoside di- and triphosphates, but only proton transport is activated by fatty acid. The detailed mechanisms of ion transport and their regulation remain unknown.</p> <p>Our approach to studying UCP independently from all other brown adipose tissue mitochondrial protein components was to functionally express UCP in a novel cellular environment. The initial goal of this project was to establish an expression system in yeast to determine whether UCP was responsible for the nucleotide-sensitive ion permeabilities observed in brown adipose tissue mitochondria. Establishing a functional yeast expression system would also allow structure/function relationships of UCP to be explored using site-directed mutagenesis of the UCP cDNA.</p> <p>An expression system for UCP in yeast was developed by inserting the rat UCP cDNA into a Saccaromyces cerevisiae/Escherichia coli shuttle vector under transcriptional control or the inducible GAL 1 promoter and transforming yeast strain JB516 (MATa, ura3, adel, leu2, his4, gal⁺) with this construct. High level expression of rat UCP in yeast mitochondria was achieved by altering the region around the start codon in the rat UCP cDNA to one resembling a highly expressed yeast gene. Expression of 70-100 μg UCP/mg mitochondrial protein is reported, a level similar to that found in brown adipose tissue mitochondria of cold adapted rats. Functional assays were developed for UCP in yeast mitochondria to measure fatty acid-activated, GDP-inhibited uncoupling of yeast mitochondria and to measure GDP bound to yeast mitochondria containing UCP. Oleate-activated uncoupling that was reversed by GDP was observed and specific for yeast mitochondria containing UCP. In addition, GDP was found to bind specifically to yeast mitochondria containing UCP with a comparable affinity to that found for brown adipose tissue mitochondria, although the level of binding was lower.</p> <p>Past chemical modification studies on UCP using cysteine and arginine modifying reagents suggested that these residues had functional significance with respect to ion transport and their regulation. However, the specific location of these residues could not be determined. Photoaffinity labelling with purine nucleotide analogs showed that the third domain of UCP was involved in purine nucleotide binding, but these studies also did not identify functionally important residues. The yeast expression system was used in conjuction with site-directed mutagenesis of the UCP cDNA to identify functionally important residues in UCP.</p> <p>Initial studies focused on the cysteines of UCP, all seven of which are conserved in UCP in all species studied to date. Each cysteine was changed independently to serine. Yeast mitochondria containing independently each mutant form of UCP showed fatty acid-activated, GDP-inhibited uncoupling. Cysteine 253 was modified further to alanine and phenylalanine to further investigate the role of this residue since cysteine 253 had been covalently labelled with a purine nucleotide analogue. These substitutions were also without effect on UCP function in yeast mitochondria. From the cysteine studies, it appears that none of the seven cysteines in UCP serve a critical role in fatty-acid activated, GDP-inhibited uncoupling. The next study investigated the role of arginine 276 in UCP. This residue was mutated to both glutamine and leucine. Yeast mitochondria containing the mutant proteins showed normal fatty acid-activated uncoupling, but the inhibitory effect of GDP was lost. Arginine 276 was therefore found to be involved in purine nucleotide regulation of UCP, but presently, it is not known if this residue is involved directly in the binding of purine nucleotides or in propogating the response in the protein to bound nucleotide.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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