The long range objectives of this project are to better understand: 1) the molecular mechanism(s) by which pyruvate (a monocarboxylate) and citrate (a tricarboxylate) are transported across the mitochondrial inner membrane; and 2) the nature of the regulation of the transport proteins catalyzing these processes (i.e., the pyruvate and tricarboxylate transport systems) by non- covalent interaction with metabolic intermediates in both normal and disease states. This proposal represents a request for support for 5 years to study the pyruvate/H+ symporter and the tricarboxylate-H+/dicarboxylate antiporter of rate liver mitochondria. These systems will be studied with respect to structure and function at the molecular/chemical levels, and with respect to regulation by physiological/pathological effectors and pharmacological agents. Specifically, experiments will be carried out to: 1) Determine the optimal conditions required for the extraction, stabilization, and functional reconstitution (in proteoliposomes) of the mitochondrial pyruvate and tricarboxylate transporters; 2) Purify these carriers to homogeneity in fully functional form; 3) Identify the type and number of amino acids within the two transporters that are essential for transport and are likely to reside within the substrate binding sites; 4) Determine the amino acid sequences of the putative substrate binding domains; and 5) Characterize the direct effect of proposed physiological/pathological regulations as well as pharmacological agents on the functioning of the purified transporters. These studies are both necessary and fundamental to our understanding of the molecular/chemical mechanism(s) by which anions are transported across biological membrane, and the potential ability of metabolic intermediates and pharmacological agents to directly regulate the functioning of anion transporters in normal and diseased states.
