Abstract
Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades these unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphological perturbations. Although structures of the bacterial and human Lon protease reveal a hexameric assembly, yeast PIM1 was speculated to form a heptameric assembly and is uniquely characterized by a ∼50-residue insertion between the ATPase and protease domains. To further understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-electron microscopy structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrates to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ∼15-residue C-terminal extension. These additional C-terminal residues form an α-helix located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1’s enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.
Highlights
An adenosine triphosphatase associated with a variety of cellular activities (AAA+) serine protease, PIM1, modulates mitochondrial DNA copy number [7] and maintains quality control and respiration [8]
Atomic structures of bacterial and human Lon define a hexameric assembly for Lon protease, initial negativestain electron microscopy and analytical centrifugation suggested that yeast Lon (PIM1) oligomerizes into a heptameric assembly [12]
PIM1 from S. cerevisiae is defined by charged insertion of approximately 50 residues between the ATPase and protease domains (Fig. S1)
Summary
Protein homeostasis (proteostasis)—the dynamic balancing of protein composition in a cell—must be tightly controlled for cellular function and longevity [1]. As the hub of bioenergetic processes, mitochondria have an array of mechanisms to maintain proteostasis, one of which comprises proteases that site-selectively cleave substrates or degrade misfolded proteins [2] These proteases acutely manage stress and prevent the accumulation of potentially toxic misfolded and aggregated proteins. Despite structural conservation of the regions that flank the 50-residue charged insertion unique to S. cerevisiae, we did not observe ordered density corresponding to the charged residues, indicating this region is independently flexible. Biochemical assays targeting this charged region demonstrate minimal effects on Lon protease function, indicating that the yeast-specific charged insertion is dispensable for its enzymatic function
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