Abstract
Yme1 is a conserved ATP-dependent protease that maintains mitochondrial function by degrading proteins in the intermembrane space. However, how Yme1 selects substrates within the crowded mitochondrial environment is poorly understood. An established substrate of Yme1 in yeast is the Tim10 subunit of the small Tim9-Tim10 protein chaperone complex, which is degraded following disruption of the subunit's internal disulfide bonds. Here, we use biochemical and biophysical approaches to examine initial substrate binding and degradation of small Tim proteins by Yme1 and shed light on the molecular mechanism of substrate selection. We show that Yme1 preferentially binds Tim10 over other small Tim proteins by forming a high-affinity interaction with the subunit irrespective of the presence of its disulfide bonds. This interaction is primarily mediated by Tim10's flexible N-terminal 'tentacle', though substrate unfolding exposes additional contact sites that enhance engagement. Notably, the human ortholog TIMM13 is also recognized by yeast Yme1, suggesting conservation of recognition strategy across species. Yme1 also binds to the assembled Tim9-Tim10 chaperone but independently of the Tim10 N-terminal tentacle. These findings suggest that Yme1 surveils the folding state of Tim10 throughout its functional lifecycle - both as a folded monomer and as a subunit of the functional chaperone complex - but only commits to degradation after disruption of its disulfide bonds.