The biophysical mechanism of protein translocation into mitochondria will be studied. Most proteins imported from the cytoplasm into mitochondria contain amino-terminal targetting sequences that direct the uptake of the proteins into the organelle. These sequences have characteristic physical properties and function as independent domains of the precursor proteins. The minimal sequence necessary for targetting the beta-subunit of the yeast F1-ATPase into mitochondria has been identified and heavily mutagenized by other workers. These studies have identified residues that are critical for the function of this targetting sequence. Peptides corresponding to the the wild-type sequence, and a series of single and double point mutants that are greatly diminished in their ability to import proteins into mithocondria in vivo, will be synthesized chemically. The physical behavior of the peptides will be measured using biophysical techniques and compared to the properties previously found for other mitochondrial targetting sequences. Sensitive and quantitative fluorescence- based assays will then be used to measure peptide binding to and import into isolated yeast mitochondria. Effects of the mutations on interactions of the peptides with mitochondria will identify features of a presequence that are important for its function. The mitochondrion is the major energy transducing unit of eukaryotic cells, containing within it the enzymatic machinery to carry out oxidative phosphorylation (i.e., oxidative conversion of C-C bond energy to CO2 and ATP). The organelle (like its cousin, the chloroplast) contains its own DNA, RNA, and protein-synthetic machinery, and some of its integral proteins are mitochondrial gene products. However (again like the chloroplast), most of the proteins found in the mitochondria are products of nuclear genes, which are translated by cytoplasmic ribosomes and subsequently translocated across the mitochondrial double membrane system. This project addresses the very important problem of the mechanism of this protein translocation process.
