Abstract
ATP-dependent proteases are ubiquitous across all kingdoms of life and are critical to the maintenance of intracellular protein quality control. The enzymatic function of these enzymes requires structural stability under conditions that may drive instability and/or loss of function in potential protein substrates. Thus, these molecular machines must demonstrate greater stability than their substrates in order to ensure continued function in essential quality control networks. We report here a role for ATP in the stabilization of the inner membrane YME1L protease. Qualitative fluorescence data derived from protein unfolding experiments with urea reveal non-standard protein unfolding behavior that is dependent on [ATP]. Using multiple fluorophore systems, stopped-flow fluorescence experiments demonstrate a depletion of the native YME1L ensemble by urea-dependent unfolding and formation of a non-native conformation. Additional stopped-flow fluorescence experiments based on nucleotide binding and unfoldase activities predict that unfolding yields significant loss of active YME1L hexamers from the starting ensemble. Taken together, these data clearly define the stress limits of an important mitochondrial protease.
Highlights
Adenosine triphosphate (ATP)-dependent proteases represent a ubiquitous class of molecular machines tasked with the maintenance of protein quality control under varied stress conditions [1,2,3,4,5]
The continued functionality of YME1L under stress conditions requires it to be stable upon acute exposure to various environmental stressors
Complementary stopped-flow fluorescence experiments reporting on YME1L unfolding based on tryptophan emissions indicate that preincubation of YME1L in the presence of saturating [ATP] yields decreased time course amplitudes relative to apo conditions, consistent with nucleotide-dependent YME1L stabilization against unfolding (Figure S2)
Summary
ATP-dependent proteases represent a ubiquitous class of molecular machines tasked with the maintenance of protein quality control under varied stress conditions [1,2,3,4,5]. These macromolecular complexes are responsible for the regulated removal of protein substrates recognized as misfolded, aggregated, or degradation-tagged [1,3,6,7]. AAA+ protease-catalyzed protein substrate unfolding and translocation requires AAA+ domain motions that are coupled to nucleotide hydrolysis [13,16,17,18] For this reason, conformational dynamics for these protease machines represent a significant driving force in dictating stress response and functional output
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