The long-term objective of this project is to elucidate the molecular structure of the mitochondrial citrate transport protein (CTP) at high resolution and to define the functions of specific amino acid residues in the translocation process. This transporter catalyzes the exchange of tricarboxylates, dicarboxylates, and phosphoenolpyruvate across the inner mitochondrial membrane, and as such is essential to the energy metabolism of eukaryotic cells. Recently, we have developed a procedure enabling the expression and purification of abundant quantities (i.e., 25 mg of purified CTP/liter E. coli culture) of functionally competent yeast mitochondrial CTP. From this foundation, we propose to initiate studies which will fundamentally advance our understanding of how this metabolically important transport protein functions at the molecular, chemical, and ultimately the atomic levels. Specifically, experiments will be carried out to: i) identify residues that are mechanistically essential and/or otherwise important for function via cysteine-scanning mutagenesis of conserved residues, followed by overexpression, purification, and functional characterization of the mutated CTPs; ii) identify amino acids that comprise the substrate translocation pathway through the CTP via probing the accessibility of cysteines (engineered into specific locations within transmembrane domains of a Cys-less CTP construct) to covalent modifying reagents; and iii) identify conditions enabling the growth of X-ray quality crystals of the CTP. In combination, these studies will provide information essential to an understanding of the chemical and structural bases for mitochondrial CTP function, and will set the stage for obtaining the first high-resolution structure of a metabolite transport protein. The health relatedness of this project concerns the central role of the CTP in the bioenergetics of eukaryotic cells. Furthermore, altered CTP function in certain disease states (e.g., diabetes, cancer) is an important aspect of the aberrant intermediary metabolism that characterizes these pathologies. Thus, an elucidation of the chemical and structural bases for substrate translocation through the CTP is essential to an understanding of the role of the CTP in energy production in both normal physiological and pathological states.
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