Engineered AAA+ Proteases Reveal Mechanisms of Degradation at the Mitochondrial Inner Membrane

Abstract

The Yme1 protease is a hexameric membrane-anchored AAA+ machine that derives energy from ATP to control proteostasis in the inner membrane and intermembrane space of mitochondria. Substrates of Yme1 include soluble, peripheral membrane, and integral membrane proteins but the mechanisms of substrate recognition and degradation remain elusive. Detailed solution studies of this class of membrane-bound AAA+ enzyme have been hampered by the requirement for an insoluble transmembrane span to drive oligomerization and thus form the ATPase sites. By replacing the transmembrane span with a soluble coiled-coil analogue, we have developed a method for driving assembly of soluble active hexameric Yme1 proteases that are fully competent for ATP-dependent protein degradation in vitro.Using these engineered enzymes with model substrates we show that Yme1 requires substrates to present an accessible peptide signal to initiate degradation and displays clear preferences in the amino acid sequence of the degradation signal. Furthermore, the protease is capable of delivering a significant unfolding force to processively unravel and translocate stable protein substrates. These principles are extended to physiological substrates of Yme1 by examining the degradation of the intermembrane-space protein transport complex Tim9/10. We demonstrate the existence of a mitochondrial degradation signal that is necessary and sufficient to target proteins for proteolysis by Yme1.