A proteomic approach to identify substrates of the AAA+ mitochondrial proteases

University Of Washington, Seattle WA

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Abstract

? DESCRIPTION (provided by applicant): Recent studies of the Parkinson's disease factors PINK1 and Parkin indicate that they play a critical role in the degradation of damaged mitochondria through a mitochondrial selective form of autophagy, termed mitophagy. This advance has fostered a greater appreciation of the importance of mitochondrial quality control to human health, and has led to a dramatic increase in research on mitophagy. However, mitophagy is only one of several mechanisms of mitochondrial quality control, and our recent work indicates that mitophagy accounts for less than half of all mitochondrial protein degradation in the model organism Drosophila. This unexpected finding suggests that non- mitophagic degradative processes are primarily responsible for mitochondrial protein quality control. We hypothesize from this and other findings that mitochondrial-resident proteases account for the majority of the protein degradative quality control that occurs in mitochondria. While genetic studies of the mitochondrial ATPase Associated with diverse cellular Activities (AAA+) family of proteases in yeast suggest that they play an important role in mitochondrial protein quality control, few of their substrates are known. Furthermore, the metazoan counterparts of these proteases have been little studied, despite the fact that mutations in the genes encoding several of them cause human disease. We propose to identify in vivo substrates of the four metazoan AAA+ mitochondrial proteases in Drosophila by using a stable isotope proteomic method to compare the half-lives of mitochondrial proteins in WT flies with those in flies that bear genetic perturbations targeting these proteases. Moreover, we will test whether these proteases degrade mitochondrial proteins that are damaged by two oft-cited mitochondrial stresses: oxidation and protein misfolding. Our work will advance the basic understanding of mitochondrial quality control by defining the biological roles of these proteases, and will provide a foundation to study the mechanisms by which mutations in the genes encoding these proteases cause disease.