Electron Cryomicroscopy Structure of a Membrane-anchored Mitochondrial AAA Protease

Abstract

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...

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

“…The recent structural model for the m-AAA protease obtained by cryo-EM has highlighted the fact that the short loops lying on the opposite side of the membrane to the AAA+ and PD are actually splayed out. This feature is absent in our structural model most likely due to a combination of limited resolution and the fact that the loop regions seem to be more flexible than the rest of the complex (Lee et al, 2011).…”

“…The recent cryo-EM data obtained for the yeast m-AAA complex (Lee et al, 2011) hint toward the possibility that the hexameric complexes might contain a 13-Å gap between the N-terminal transmembrane segment and the AAA+ domain through which unfolded, but not An isomesh rendered molecular envelope, contoured with a threshold of 2.5 s for the final calculated asymmetric three-dimensional map, is shown in white. The modeling in of the crystallography-derived hexameric soluble fragment of apoFtsH from T. maritima (3KDS.pdb) was done by visual inspection with atoms colored as a spectral rainbow from the N terminus (blue) to the C terminus (red).…”

“…The central core of the complex has a maximum diameter of 120 Å, close to the value of 130 Å determined by cryo-EM for the m-AAA protease (Lee et al, 2011). By assuming a protein density of 0.844 Å 3 per Dalton (van Heel et al, 1996), masses of 360 kD could be assigned to the central portion containing the AAA module and PD, 36 kD to the N-terminal cap consisting of the transmembrane helices and lumenal loops, 18 kD to the three outlying domains assigned to the 27-kD GST tag, leaving a C-terminal bottom region of 30 kD ( Figure 7D) likely to contain the C-terminal region of FtsH, which is relatively disordered and not present in the T. maritima crystal structure used in the fitting, plus possibly some of the GST tag.…”

“…Information on the structure of intact FtsH complexes, rather than Escherichia coli-expressed soluble fragments, is sparse, and it is only recently that a hexameric structure was confirmed for the heteromeric m-AAA complex of the yeast mitochondrial inner membrane by cryo-electron microscopy (cryo-EM) (Lee et al, 2011). The resolution of the structure, determined at 12 Å, was sufficient to confirm the presence of six FtsH protomers within each complex, obtained by fitting the atomic structures of cytosolic fragments of FtsH into the cryo-EM model (Bieniossek et al, 2006(Bieniossek et al, , 2009, but was insufficient to determine the structural organization of the two types of subunit within the ring.…”

Quality Control of Mitochondrial Proteostasis

“…The two-protease complexes expose their catalytic domains to opposing faces of the mitochondrial IM with the m-AAA protease exerting its activity in the mitochondrial matrix and the i-AAA protease being active in the IMS (Leonhard et al 2000). First structural information for a eukaryotic AAA þ protease was recently obtained for the yeast m-AAA protease using single particle electron cryomicroscopy (Lee et al 2010). The m-AAA protease forms a hexamer in solution, resulting in a large, ringlike molecular machine, with a central pore for substrates to enter into a proteolytic chamber.…”