Summary Ring-shaped AAA+ ATPases control a variety of cellular processes by substrate unfolding and remodeling of macromolecular structures. However, how ATP hydrolysis within AAA+ rings is regulated and coupled to mechanical work is poorly understood. Here, we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes, conserved AAA+ machines in the inner membrane of mitochondria. ATP binding to one AAA subunit inhibits ATP hydrolysis by the neighboring subunit leading to coordinated rather than stochastic ATP hydrolysis within the AAA ring. Unbiased genetic screens define an intersubunit signaling pathway involving conserved AAA motifs and reveal an intimate coupling of ATPase activities to central AAA pore loops. Coordinated ATP hydrolysis between adjacent subunits is required for membrane dislocation of substrates but not for substrate processing. These findings provide new insight how AAA+ proteins convert energy derived from ATP hydrolysis into mechanical work.

Maturation of cytochrome c peroxidase (Ccp1) in mitochondria occurs by the subsequent action of two conserved proteases in the inner membrane: the m-AAA protease, an ATP-dependent protease degrading misfolded proteins and mediating protein processing, and the rhomboid protease Pcp1, an intramembrane cleaving peptidase. Neither the determinants preventing complete proteolysis of certain substrates by the m-AAA protease, nor the obligatory requirement of the m-AAA protease for rhomboid cleavage is currently understood. Here, we describe an intimate and unexpected functional interplay of both proteases. The m-AAA protease mediates the ATPdependent membrane dislocation of Ccp1 independent of its proteolytic activity. It thereby ensures the correct positioning of Ccp1 within the membrane bilayer allowing intramembrane cleavage by rhomboid. Decreasing the hydrophobicity of the Ccp1 transmembrane segment facilitates its dislocation from the membrane and renders rhomboid cleavage m-AAA protease-independent. These findings reveal for the first time a non-proteolytic function of the m-AAA protease during mitochondrial biogenesis and rationalise the requirement of a preceding step for intramembrane cleavage by rhomboid.

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo-and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo-and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides.These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.Energy-dependent proteases form oligomeric ring complexes and harbor conserved ATPase domains of the AAA ϩ family (1). It is widely accepted that AAA ϩ machines utilize the energy derived from ATP hydrolysis to thread substrate proteins through a central pore resulting in substrate unfolding. FtsH-related AAA proteases form a distinct membraneassociated group of AAA ϩ machines, present in eubacteria and in mitochondria and chloroplasts of eukaryotic cells (2, 3). Members of this group feature an N-terminal membrane targeting signal, followed by one or two transmembrane helices, and a canonical AAA domain that is covalently linked to the metallopeptidase domain (3, 4).X-ray crystal structures of the soluble cytosolic domains of the bacterial AAA protease FtsH (FtsH Cyt ) bound to ADP and in the absence of nucleotide (apo) have been reported (5-7). Although the AAA ring of the apo-FtsH Cyt hexamer was 6-fold symmetric (7), the ADP-bound structures revealed a 2-(6) and 3-fold symmetrical hexamer (5). However, the protease ring was 6-fold symmetric in all three structures. Therefore, it remained unclear what symmetry the full-length protease adopts and which of the stereo-specific interactions between ne...

Mitochondria harbor a conserved proteolytic system that mediates the complete degradation of organellar proteins. ATP-dependent proteases, like a Lon protease in the matrix space and m-and i-AAA proteases in the inner membrane, degrade malfolded proteins within mitochondria and thereby protect the cell against mitochondrial damage. Proteolytic breakdown products include peptides and free amino acids, which are constantly released from mitochondria. It remained unclear, however, whether the turnover of malfolded proteins involves only ATP-dependent proteases or also oligopeptidases within mitochondria. Here we describe the identification of Mop112, a novel metallopeptidase of the pitrilysin family M16 localized in the intermembrane space of yeast mitochondria. This peptidase exerts important functions for the maintenance of the respiratory competence of the cells that overlap with the i-AAA protease. Deletion of MOP112 did not affect the stability of misfolded proteins in mitochondria, but resulted in an increased release from the organelle of peptides, generated upon proteolysis of mitochondrial proteins. We find that the previously described metallopeptidase saccharolysin (or Prd1) exerts a similar function in the intermembrane space. The identification of peptides released from peptidase-deficient mitochondria by mass spectrometry indicates a dual function of Mop112 and saccharolysin: they degrade peptides generated upon proteolysis of proteins both in the intermembrane and matrix space and presequence peptides cleaved off by specific processing peptidases in both compartments. These results suggest that the turnover of mitochondrial proteins is mediated by the sequential action of ATP-dependent proteases and oligopeptidases, some of them localized in the intermembrane space.Mitochondria are essential organelles with central anabolic and catabolic functions. To maintain their homeostasis and thereby avoid cell damage, a precise control of the steady state levels of mitochondrial proteins is required. First, evidence for the presence of an independent proteolytic system within mitochondria came from early studies that revealed different turnover rates of proteins residing in different mitochondrial subcompartments (1, 2). Many components of this system, often highly conserved throughout evolution, have been identified since then and found to exert crucial functions within mitochondria (3, 4). They control distinct steps in the biogenesis of mitochondria and selectively degrade misfolded and nonassembled polypeptides accumulating in the organelle. These could be non-assembled proteins, which accumulate in case of an imperfect coordination of nuclear and mitochondrial gene expression, or oxidatively damaged proteins progressively generated in aging cells. Quantitative measurements of mitochondrial protein turnover in logarithmically growing yeast cells suggested the degradation of up to 10% of the mitochondrial proteome per hour, most likely reflecting to a large extent misfolded or damaged proteins (5).Central c...