Two specific objectives will be pursued during the proposed project period. The first will be expression of the full length clone, pCGS11N, which contains the coding information for avian liver glutamine synthetase, in expression vectors such as pKK223 or various derivatives thereof. Of special concern will be a characterization of the expression product, especially its conformation. Preliminary results indicate the expression product may aggregate. Since this is likely to render it import- incompetent, conditions that prevent aggregation and also keep the protein in an unfolded configuration will be established. The second major objective will be to establish an in vitro system in which glutamine synthetase is imported into isolated mitochondria. If necessary, the original technique of presenting isolated mitochondria with freshly translated synthetase will be used, although the use of the pure expression product is now preferable and is required to establish species- and tissue-specific cytosolic import factors. %%% Seventy-five percent of land vertebrates utilize a uricotelic-type mechanism for detoxifying ammonia formed during hepatic amino acid catabolism. Ammonia, formed via the glutamate dehydrogenase reaction, is converted by glutamine synthetase to the amide function of glutamine which then exits to the cytosol where it is utilized for uric acid synthesis. In the ureotelic mechanism, utilized by mammals and some adult amphibians, ammonia is converted to citrulline in the mitochondria, which then exits to the cytosol to be converted to urea. What these two pathways have in common is that in each case, the ammonia-detoxifying mechanisms are directed toward converting ammonia formed in the mitochondrial matrix to a form that does not bind protons during transit of the inner mitochondrial membrane and therefore does not uncouple oxidative phosphorylation. What is particularly interesting is that, in ureotelic vertebrates (e.g., mammals), the enzyme glutamine synthetase is only found in the cytosol, never in the mitochondria, whereas in the uricotelic sauropsid vertebrates (reptiles and birds) the enzyme is localized to mitochondria in the liver but is cytosolic in other tissues such as neural tissue. A putative mitochondrial targeting sequence in the avian enzyme, not present in the mammalian enzyme, has been identified. However, the primary amino acid sequences of the avian liver (mitochondrial) and neural tissue (cytosolic) forms are identical, raising the question of how the differential targeting occurs. This project aims to understand the molecular events responsible for targeting of the enzyme in sauropsids. The work is of considerable interest from biochemical, cell biological, physiological, and evolutionary perspectives.
