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Free keywords:
ATP-Dependent Proteases
Adenosine Triphosphatases/chemistry/*genetics/*physiology
Amino Acid Motifs
Amino Acid Sequence
Binding Sites
Cloning, Molecular
Fungal Proteins/chemistry/genetics/metabolism
Hydrolysis
Intracellular Membranes/*enzymology
Mitochondria/enzymology/metabolism
*Point Mutation
Precipitin Tests
Prostaglandin-Endoperoxide Synthases/genetics/metabolism
Protein Binding
Protein Folding
Protein Processing, Post-Translational
Saccharomyces cerevisiae/genetics/metabolism
Saccharomyces cerevisiae Proteins/chemistry/*genetics/*physiology
Substrate Specificity
Tetrahydrofolate Dehydrogenase/genetics/metabolism
Abstract:
Two membrane-bound ATP-dependent AAA proteases conduct protein quality surveillance in the inner membrane of mitochondria and control crucial steps during mitochondrial biogenesis. AAA domains of proteolytic subunits are critical for the recognition of non-native membrane proteins which are extracted from the membrane bilayer for proteolysis. Here, we have analysed the role of the conserved loop motif YVG, which has been localized to the central pore in other hexameric AAA(+) ring complexes, for the degradation of membrane proteins by the i-AAA protease Yme1. Proteolytic activity was found to depend on the presence of hydrophobic amino acid residues at position 354 within the pore loop of Yme1. Mutations affected proteolysis in a substrate-specific manner: whereas the degradation of misfolded membrane proteins was impaired at a post-binding step, folded substrate proteins did not interact with mutant Yme1. This reflects most likely deficiencies in the ATP-dependent unfolding of substrate proteins, since we observed similar effects for ATPase-deficient Yme1 mutants. Our findings therefore suggest an essential function of the central pore loop for the ATP-dependent translocation of membrane proteins into a proteolytic cavity formed by AAA proteases.
