The mitochondrial myopathies include neuromuscular disorders that result from defects in the assembly and/or function of various mitochondrial enzyme complexes. These disorders affect the central nervous system, skeletal muscle, heart, kidney and liver. In some of these diseases the severity of clinical symptoms is directly proportional to the extent of the defects in oxidative phosphorylation. The goal of this project is to understand how defects in this process could produce musculoskeletal disease. We initially intend to approach this objective by addressing the molecular mechanisms involved in the biogenesis of a single mitochondrial enzyme complex, the proton-translocating ATPase of the yeast Saccharomyces cerevisiae. In this proposal, I will describe experiments designed to increase our understanding of the processes important for import of the subunits of the F1-ATPase are all encoded in the nuclear genome, translated in the cytosol, and imported into mitochondria in a post-translational manner. In order to better understand F1-ATPase biogenesis, we must first recognize the processes that allow it to be efficiently imported into mitochondria. While many general features of mitochondrial protein import have been elucidated, many of the specific factors and interactions that facilitate this process remain largely uncharacterized. In previous studies we isolated and characterized a large number of deletion and missense mutations in the mitochondrial import signal of the F1-ATPase beta subunit precursor. Through the analysis of these mutant proteins we demonstrated that this targeting signal contains functionally redundant targeting information and found a strong correlation between the efficiency of mitochondrial protein import and the ability of a """"""""minimal targeting signal"""""""" to assume an amphophilic alpha helical conformation. More recently, we obtained evidence suggesting that some of these mutant targeting signals are unable to maintain the precursor in an import competent conformation. In light of these results, we intend to characterize the specific cytosolic factors and interactions necessary for efficient mitochondrial import of the beta subunit precursor and determine how mutations within the beta subunit mitochondrial import signal may alter these associations. By better understanding the processes leading to the proper subcellular localization of the subunits of this enzyme complex, we will increase our knowledge of the general mechanisms involved in mitochondrial biogenesis.
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