Abstract
Mitochondrial AAA proteases harness energy from ATP hydrolysis to remove and degrade damaged protein components of the mitochondrial inner membrane. Transmembrane domains anchor the proteases in the inner membrane and drive assembly of the active hexameric form of the enzymes. To date, the requirement for these transmembrane domains has hampered detailed analysis of the substrate binding and processing activities of these important enzymes. The objective of this study is to develop a system for reconstituting membrane‐anchored ATP‐dependent proteases in a soluble, assembled, and active form that can be used to elucidate the molecular mechanisms of these important enzymes.We report a novel method for reconstructing soluble, assembled and active proteases by replacing the insoluble transmembrane domain with a soluble analogue. In combination with a series of solution biochemical assays, we demonstrate that the human mitochondrial AAA protease, YME1L, is a capable of mechanically unfolding stable protein substrates, requires substrates to contain a recognition sequence of defined sequence, and consumes substrates by processive unfolding and translocation. Together these results clarify a number of open questions in the field and provide a framework for the study of the wider family of membrane‐anchored AAA+ molecular machines.This research was supported by Stony Brook University.
Published Version
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