Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease

Abstract

The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate.

Unique structural features of the mitochondrial AAA+ protease AFG3L2 reveal the molecular basis for activity in health and disease

“…To verify the crucial role of F381 in substrate translocation, we used established assays to monitor ATP hydrolysis and substrate degradation for an engineered hexameric AFG3L2, where the transmembrane domains were substituted by a hexamerizing coiled coil ( cchex AFG3L2) 42 ( Figure 1B). As previously shown, the coiled coil ensures hexamerization of active subunits in the absence of any stabilizing mutations 43,44 . Incorporation of an F381A substitution abolished substrate degradation, while only mildly impacting ATP hydrolysis ( Figure 2B), confirming that the aromatic pore-loop 1 residue in AFG3L2 plays a conserved, essential role in substrate translocation.…”

“…ATPase activity, fluorescence-based protein degradation, and fluorogenic peptide cleavage assays were performed as previously described with some modifications 42,44 . ATPase assays were carried out at 37 • C in a 384-well clear bottom plate (Corning) using a SpectraMax M5 plate reader (Molecular Devices) with 1 µM cchex AFG3L2 or its variants.…”

Atomic structure of the mitochondrial inner membrane AAA+ protease YME1 reveals the mechanism of substrate processing

“…To confirm that these observed pore-loop interactions play a role in substrate translocation, we examined the substrate degradation activity of hex YME1 containing point mutations in the pore-loop 1 tyrosine (Y354A) or the pore-loop 2 tyrosine (Y396A). Introduction of these mutations into either pore loop largely abolish degradation of the I27 domain of human titin bearing an N-terminal degron derived from the mitochondrial Tim10 protein (T10-I27) ( Figure 2B) (Rampello and Glynn 2017). The introduction of mutations in the I27 domain to destabilize the folded structure (T10-I27 CD ) produced a large increase in degradation rate by wild-type hex YME1, likely resulting from the removal of the unfolding step of degradation (Kenniston 2003).…”

“…All additional variants of hex YME1 were generated by site-directed mutagenesis using wild-type hex YME1 as a template. All hex YME1 variants were expressed and purified as previously described (Shi 2016, Rampello andGlynn 2017) with the following alterations. Proteins used in cryo-EM studies were buffer exchanged immediately prior to size exclusion chromatography into a buffer containing 20mM Tris-HCl (pH 8.0), 300mM NaCl, 2mM EDTA, 10% glycerol, and 1mM DTT.…”

“…A recently engineered soluble construct of yeast YME1, hex YME1, comprising the AAA+ ATPase and protease domains assembled into active oligomers by fusion to a hexamerizing domain, exhibits high ATPase and proteolytic activity, as well as biologically relevant substrate specificity (Shi 2016, Rampello andGlynn 2017). Here, we employed a hex YME1 variant containing an E381Q Walker B mutant ( hex YME1 WB ) that limits ATP hydrolysis to enable structural determination of this ATP-dependent protease by cryo-electron microscopy (cryo-EM) (Figure 1, Supplementary Figures 1-2).…”