The long-range objective of this project is to understand, the structure and function of the mitochondrial tricarboxylate transport protein. This protein catalyzes a tricarboxylate- H+/dicarboxylate antiport across the mitochondrial inner membrane, an action which is essential to the energy metabolism of the liver parenchymal cell. Experiments will be conducted to: 1) isolate, characterize and sequence the cDNA that encodes the entire rat liver mitochondrial CTP in order to deduce the complete amino acid sequence of this carrier; 2) elucidate the topography of the CTP within the mitochondrial inner membrane via the use of site- specific polyclonal antibodies, in situ proteolysis, and vectorial labeling; and 3) begin to identify and localize specific amino acid residues which form the substrate binding site within the CTP through the use of both active-site directed and group-directed probes in combination with amino acid sequence analysis of modified domains. The proposed studies are both necessary and fundamental for an in depth understanding of the molecular mechanisms that biological systems utilize to transport anions across membranes and thereby meet metabolic meeds. %%% The aim of this project is to understand, at the molecular and chemical levels, the structure and function of the mitochondrial citrate transport protein (CTP). This protein transports citrate and malate across the mitochondrial inner membrane and is essential to the energy metabolism of hepatic cells. In order to accomplish this aim we will conduct experiments to: 1) deduce the complete amino acid sequence of the rat liver mitochondrial CTP; 2) determine how this transport protein is folded and orientated within the mitochondrial inner membrane; and 3) begin to identify and localize specific amino acid residues which are essential to the transport mechanism and which form the citrate and malate binding site(s). These studies are fundamental to an in depth understanding of the molecular mechanisms that biological systems utilize to transport molecules across membranes to meet their metabolic needs. More generally, these investigations are expected to provide a significant advance in our knowledge about how transport proteins work.
