9316810 Roise This project will focus on an effort to apply quantitative fluorescence techniques to tde study of the import of a full- length mitochondrial precursor protein into isolated yeast mitochondria. These studies will provide quantitative information about binding and import that is not currently available using radiolabeled precursor proteins. The approaches may also be useful in the characterization of import intermediates that span the mitochondrial membranes. The precursor under study is a fusion between the amino- terminal 25-residue mitochondrial targeting sequence of yeast cytochrome oxidase subunit IV (CoxIV) and the complete amino acid sequence of mouse dihydrofolate reductase (DHFR). The pCoxIV-DHFR fusion protein is efficiently imported by mitochondria both in vitro and in vivo and has been used extensively to study the import process. The protein has recently been labeled with the NBD fluorophore specifically attached to cysteine-19 of the presequence. The labeled precursor can now be used in studies similar to those developed with the fluorescent presequence peptide. In addition, the laboratory will test whether or not the full- length precursor can be imported in Paracoccus denitrificans, a gram-negative bacterium that is typically used as a model for the ancestral mitochondrion and which has recently been shown to be capable of importing a synthetic mitochondrial presequence. This ability would be important in the context of understanding the evolution of the protein transport system in the mitochondria of modern eukaryotic cells. The results obtained in the import experiments with mitochondria will be used to develop optimal conditions to look for import with the bacteria. Even if the precursor is not fully translocated, the fluorescent approaches should provide information about the extent and type of interaction of the precursor protein with these bacteria. %%% Eukaryotic cells are believed to have evolved from endosym biotic relationships between certain protistan "host" cells and other "endosymbiont" protists. Modern eukaryotes contain specialized organelles (mitochondria and plastids) which still retain substantial vestiges of autonomy (genomes and protein-synthesizing machinery). The successful function of a modern eukaryotic cell hinges on the interdependence and appropriate exchange of proteins and other information between these organelles and the the rest of the cell (nucleus and cytoplasm). This project addresses not only the mechanism whereby nuclear-encoded proteins get into the mitochondrion in order to carry out their proper functions, but also begins to address, at the biochemical and molecular levels, the question of how this mechanism may have evolved. The kind of information that is expected to come from such studies will be valuable in biotechnological applications that require specific manipulation of transport phenomena involving proteins inserting into or crossing membrane barriers. ***
