STRUCTURE/FUNCTION OF MITOTARGETING SIGNAL SEQUENCES

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DESCRIPTION: Mitochondrial matrix space proteins are coded by nuclear genes and translocated into the organelle after their synthesis. An N-terminal extension of polypeptide, called a presequence, allows the precursor protein to recognize mitochondria and be trannnslocated across the two membranes in an energy-dependent process. After import, the presequence is removed by proteolysis for most, but not all precursor proteins. There is no homologous amino acid sequence among mitochondrial presequences that can be shown to give rise to their targeting properties. There is little experimental data available that defines the sequence or structural requirements for the processing site. The major similarities in mitochondrial presequences are a net positive charge and the predicted capability to form amphiphilic structures. It has previously been determined, using two dimensional NMR, circular dichroism and fluorescence techniques, that synthetic peptides corresponding to mitochondrial presequences form amphiphilic alpha helices. Preliminary data suggests that the ability of the presequence to bind to the mitochondrial membrane and the helical stability at the N-terminus are crucial features of mitochondrial targeting. In addition, a model has been developed for the requirements of processing. The model employs a combination of the primary and secondary structure of presequences as a recognition motif for the processing protease. Further understanding of the structural requirements for presequences to bind to mitochondria, be translocated into the matrix and be processed by the protease will be gained by making mutations in them. Import rates and mitochondrial binding properties of proteins containing the mutated presequences will be measured. The success of a mutant presequence in carrying out these functions will be compared to biophysical data regarding structure and membrane binding of the related synthetic peptide. The goal is to determine the contribution that helicity, charge and hydrophobicity make to the binding import and processing of various presequences. To further realize this goal, import will be performed in transformed yeast and HeLa cells to verify that the conclusions based on in vitro studies are consistent with import in vivo. As a result, thorough knowledge of proteins are targeted to mitochondria and how to prevent protein import will be obtained. This knowledge could be used to target theraputic agents to mitochondria. One such strategy could involve the import of a protein that is normally encoded by a mitochondrial gene, when that gene has been damaged. With a thorough understanding of import, it may become possible to restore mitochondrial function to people afflicted by genetic defects in mitochondria-encoded proteins. In addition, it is conceivable that organic analogs could be designed to act as antagonists and prevent import into mitochondria.