This proposal will investigate the mechanism by which mitochondrial precursor proteins synthesized in the cytoplasm are subsequently localized to mitochondria. The model for this post-translational uptake is the biosynthesis of rat mitochondrial malate dehydrogenase and glutamate dehydrogenase. Both of these proteins are synthesized as larger precursors which are proteolytically processed to their mature sizes during translocation into mitochondria. cDNA clones encoding all or part of these protein precursors have been isolated. These will be used to determine the structures of the precursor transit peptides by nucleotide sequence analysis. Mutations will be introduced into the transit peptides of both proteins through deletion and site-directed mutagenesis. Both normal (wild-type) and mutant cDNAs will be transcribed in vitro into the corresponding mRNAs using the SP64 system. mRNA translation in vitro and incorporation into isolated mitochondria in vitro will determine the effects of the mutations on binding, uptake and proteolytic processing of the precursors. We will also incorporate the cDNAs encoding the precursors into a bacterial expression vector in order to generate pure precursor and mutant proteins in large amounts. An attempt to compare the x-ray crystallographic structure of the precursor with the mature protein will be made. The precursor protein will be used to examine the kinetics of binding to the mitochondrial receptor and, possibly, to begin its purification. Finally, genomic clones for pre-mMDH and pre-GDH will be isolated and the structure analyzed.
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