Mechanistic Investigation of Proteostasis at the Outer Mitochondrial Membrane

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Mechanism of Msp1 Mediated Protein Extraction from the Mitochondrial Membrane As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain homeostasis of the proteome (proteostasis). Declines in mitochondrial function and/or proteostasis have been linked to cancer, aging, cardiovascular, and neurodegenerative diseases. A poorly understood aspect of mitochondrial proteostasis is the removal of membrane proteins from the lipid bilayer. This is due to the technical challenges of reconstituting this process in vitro. We have overcome this technical barrier by developing a simple, but powerful reconstituted system with the AAA+ (ATPase Associated with cellular Activities) protein Msp1. Anchored in the outer mitochondrial membrane (OMM), Msp1 maintains mitochondrial proteostasis by removing unwanted proteins from the lipid bilayer. Mutations in Msp1 or the human homologue ATAD1 lead to compromised mitochondrial function, impaired fear conditioning, severe encephalopathy, and early death. Despite its clear physiological importance, there are many important unanswered questions regarding Msp1 activity. How does Msp1 interact with other quality control components to maintain membrane proteostasis? What is the full range of substrates extracted by Msp1/ATAD1? How is this regulated? These are particularly pressing questions given that our collaborator, Jared Rutter (HHMI, University of Utah), has preliminary genetic evidence that ATAD1 may regulate apoptosis by extracting BH3-only proteins from the OMM. We will use an unbiased proteomic approach and our in vitro extraction assay to directly test this paradigm shifting hypothesis and examine the molecular details for how this process is regulated. Because our reconstituted system overcomes key technical barriers that have hampered previous attempts to study the extraction of membrane proteins from a lipid bilayer, we will also utilize our system to draw foundational conclusions about how key factors such as membrane fluidity, substrate structure, and ATP hydrolysis rates affect this essential cellular process. This work will test an exciting new hypothesis for apoptotic regulation, provide a comprehensive picture of Msp1/ATAD1 function in mitochondrial biology, and uncover new insights into the fundamental process of membrane protein extraction.