The mitochondrial inner membrane is the site of essential cellular functions such as oxidative phosphorylation, phospholipid metabolism, and the regulation of apoptosis. These activities are performed by a composite mitochondrial proteome that requires constant resculpting to respond to both the changing metabolic demands of the cell and the emergence of damage driven by reactive oxygen species. This resculpting is performed by two mitochondrial AAA+ proteases, which harness the energy of ATP to recognize, unfold and degrade protein substrates both from within and surrounding the inner membrane. In humans, dysfunction of these proteases has been linked to the development of severe neurodegenerative disorders such as spinocerebellar ataxia. AAA+ proteases assemble as hexamers to form an internal proteolytic chamber into which substrates are forcibly translocated by a ring of ATPases. The study of the mitochondrial AAA+ proteases has been long hampered by their combination of multiple soluble catalytic domains with insoluble transmembrane domains for anchoring into the inner membrane. We utilize a protein- engineering approach to assemble previously membrane-constrained hexameric proteases in a soluble, active form. Our goal is to use these rebuilt proteases to perform a rigorous analysis of the mechanisms driving energy-dependent proteolysis at the mitochondrial inner membrane. The first aim of the proposal is to define how substrates are selected for degradation among the myriad mitochondrial proteins. Degradation signal sequences will be identified from physiological substrates to ask whether these signals are conserved across diverse mitochondrial proteins to enable recognition by common proteases. The second aim is to examine the recognition complex formed between these proteases and specific substrates. A series of complementary biochemical approaches will map the protease substrate binding sites and identify the complementary contacts used to promote selection and degradation. Finally, we will examine how the architecture of the proteolytic sites within the degradation chamber achieves specificity of peptide-bond cleavage specificity, resulting in site-specific cleavage of a class of substrates, including the regulator of mitochondrial fission. Together, these experiments will provide a rigorous mechanistic analysis of the mitochondrial AAA+ proteases and provide foundational knowledge to aid the development of small molecule modulators as future therapeutics.