Molecular Analysis of Plant Mitochondrial Substrate Transporters

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A unique group of monoclonal antibodies that are specific for the dicarboxylate, monocarboxylate, and glutamate/aspartate transporters of the inner mitochondrial membrane of pea plants will be used to screen lambda gt11 libraries for cDNA clones of these transport proteins. A partial clone for the dicarboxylate transporter has already been isolated and the work will initially concentrate on this protein. Once full length clones are available, they will be sequenced and molecular modelling techniques used to evaluate their structures within the membrane. When a structural model has been obtained, it will be tested by using antibodies and chemical modifiers, both targeted to synthetic peptides, to confirm the model by evaluating that portions of the proteins are exposed to the cytosolic and matrix face of the mitochondrial membrane. The cDNA clones will be used to produce the precursor form of the protein by in vitro transcription/translation and heterologous expression in bacteria. These immature forms of the protein will be used to study the incorporation of the transporter into the mitochondrial membrane. They also will be used in an attempt to isolate and reconstitute the receptor protein that is responsible for recognizing the transport protein and guiding its incorporation into the membrane. Mitochondria are the living cell's energy factory. Movement of molecules into and out of mitochondria plays an integral part in the cell's energy metabolism. The mitochondrion has a very complex membrane structure which has not been amendable to biochemical analysis. New techniques of molecular biology are being used to understand the membranes in which energy is generated and molecules are transported.