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.

Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines.

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...