Lipid bilayer properties govern substrate engagement and extraction by the AAA+ ATPase Msp1

An essential aspect of protein quality control is enzymatic removal of membrane proteins from the lipid bilayer. Failures in this critical cellular process are associated with neurodegenerative diseases and cancer. Msp1 is a AAA+ (ATPases associated with diverse cellular activities) ATPase that removes mistargeted membrane proteins from the outer mitochondrial membrane. How Msp1 selectively recognizes and extracts substrates within the complex outer mitochondrial membrane ecosystem, and how the lipid bilayer impacts these processes are unknown. Here, we describe the development of a fully defined, rapid, and quantitative extraction assay that retains physiological substrate selectivity. Using this new assay, we systematically modified both the model substrate and the lipid environment to demonstrate that Msp1 can recognize substrates by a hydrophobic mismatch between the substrate transmembrane domain and the lipid bilayer. We further demonstrate that the rate-limiting step in Msp1 activity is extraction of the transmembrane domain from the lipid bilayer. Together, these results provide foundational insights into how the lipid bilayer influences AAA+ mediated membrane protein extraction.