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3.A.29.  The Mitochondrial Inner Membrane i-AAA Protease Complex (MIMP) Familly 

The mitochondrial inner membrane i-AAA protease supercomplex is required for mitochondrial inner membrane protein insertion, degradation and turnover. It is important to maintain the integrity of the mitochondrial compartment and is required both for the degradation of unassembled subunit 2 of cytochrome c oxidase (COX2) and for efficient assembly of the mitochondrial respiratory chain. It binds unfolded substrates in an ATPase-independent manner. Binding of folded COX2, a physiological substrate, requires an active ATPase, but when COX2 is destabilized, an active ATPase is no longer necessary.  Central pore mutants of Yta11 (Yme1, OSD1) have been isoated (Graef and Langer 2006; Dunn et al. 2006).  Family 9.B.307 was transfered to Family 3.A.29, and protein entries 9.B.307.1.1, 1.2 and 1.3 became proteins 3.A.29.1.4, 1.5 and 1.6.

Precursors of β-barrel and α-helical proteins are transported into the outer membrane via distinct import routes. The translocase of the outer membrane (TOM complex; TC# 3.A.8) transports β-barrel precursors across the outer membrane, and the sorting and assembly machinery (SAM complex; also 3.A.8) inserts them into the target membrane. The mitochondrial import (MIMP) complex (this family) constitutes the major integration site for alpha-helical embedded proteins. The import of some MIM-substrates involves TOM receptors, while others are imported in a TOM-independent manner. Thus, TOM, SAM and MIM complexes dynamically interact to import a large set of different proteins and to coordinate their assembly into protein complexes (Gupta and Becker 2020).

Protein complexes involved in respiration, ATP synthesis, and protein import reside in the mitochondrial inner membrane. The m-AAA protease, a conserved hetero-hexameric AAA (ATPase associated with diverse cellular activities) protease, composed of the Yta10, Yta11 and Yta12 proteins, regulates mitochondrial proteostasis by mediating protein maturation and degradation. It also recognizes and mediates the dislocation of membrane-embedded substrates, including foreign transmembrane (TM) segments. The Yta10 TMS2 domain is essential for membrane dislocation for only a subset of substrates, whereas the Yta12 TMS2 domain is essential for membrane dislocation for all tested substrates, suggesting different roles of the TM domains in these two m-AAA protease subunits. m-AAA protease-mediated membrane dislocation was impaired in the presence of a large downstream hydrophilic moiety in a membrane substrate, suggesting that the m-AAA protease cannot dislocate large hydrophilic domains across the membrane.  Thus, membrane dislocation depends on the TMSs (Lee et al. 2017).

Cellular proteomes are dynamic and adjusted to permanently changing conditions by ATP-fueled proteolytic machineries. Among the five AAA+ proteases in Escherichia coli, FtsH is the only essential membrane-anchored metalloprotease. FtsH is a homohexamer that uses its ATPase domain to unfold and translocate substrates that are subsequently degraded without the need of ATP in the proteolytic chamber of the protease domain (Bittner et al. 2017). FtsH eliminates misfolded proteins in the context of general quality control and properly folded proteins for regulatory reasons. Trapping approaches have revealed a number of novel FtsH substrates. The review by Bittner et al. 2017 summarizes the substrate diversity of FtsH and presents details on the diverse recognition principles of three well-characterized substrates: LpxC, the key enzyme of lipopolysaccharide biosynthesis; RpoH, the alternative heat-shock sigma factor and YfgM, a bifunctional membrane protein implicated in periplasmic chaperone functions and cytoplasmic stress adaptation.

Crystal structures of a transmembrane helix-lacking FtsH construct from Aquifex aeolicus have been determined at 2.9 Å (Uthoff and Baumann 2018). The FtsH hexamer is created from two different subunits of the asymmetric unit by the three-fold symmetry of the crystals. Similar to other published structures, all subunits are loaded with ADP, and the two subunits in the asymmetric unit resemble the already known open and closed conformations. Within the ATPase cycle, while the whole subunit switches from the opened to the closed state, pore loop-1 interacts with the substrate and translocates it into the proteolytic chamber. An inactive conformation of the pore loop allows the closed conformation to switch back to the opened state without pushing the substrate out again. This reveals how this new pore loop conformation is induced and how it is linked to the intersubunit signalling network (Uthoff and Baumann 2018).

AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore towards a degradation chamber. FtsH, a membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. Fully active FtsH dodecamers consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer, visualized by negative stain TEM, suggests lateral substrate entry between membrane and cytosolic domains. Such a substrate path was resolved in the cryo-EM structure of the FtsH hexamer (Carvalho et al. 2020). By mapping the available structural information and structure predictions for the TMSs to the amino acid sequence, a linker of ~20 residues between the second TMS and the cytosolic domain was identiied. This unique polypeptide appears to be highly flexible, and turned out to be essential for proper functioning of FtsH (Carvalho et al. 2020).






This family belongs to the: AAA-ATPase Superfamily.

References associated with 3.A.29 family:

Bittner, L.M., J. Arends, and F. Narberhaus. (2017). When, how and why? Regulated proteolysis by the essential FtsH protease in Escherichia coli. Biol Chem 398: 625-635. 28085670
Carvalho, V., I. Prabudiansyah, L. Kovacik, M. Chami, R. Kieffer, R. van der Valk, N. de Lange, A. Engel, and M.E. Aubin-Tam. (2020). The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry. J. Biol. Chem. 296: 100029. [Epub: Ahead of Print] 33460938
Carvalho, V., I. Prabudiansyah, L. Kovacik, M. Chami, R. Kieffer, R. van der Valk, N. de Lange, A. Engel, and M.E. Aubin-Tam. (2020). The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry. J. Biol. Chem. [Epub: Ahead of Print] 33154162
Dunn, C.D., M.S. Lee, F.A. Spencer, and R.E. Jensen. (2006). A genomewide screen for petite-negative yeast strains yields a new subunit of the i-AAA protease complex. Mol. Biol. Cell 17: 213-226. 16267274
Graef, M. and T. Langer. (2006). Substrate specific consequences of central pore mutations in the i-AAA protease Yme1 on substrate engagement. J Struct Biol 156: 101-108. 16527490
Gupta, A. and T. Becker. (2020). Mechanisms and pathways of mitochondrial outer membrane protein biogenesis. Biochim. Biophys. Acta. Bioenerg 1862: 148323. [Epub: Ahead of Print] 33035511
Lee, S., H. Lee, S. Yoo, and H. Kim. (2017). Molecular insights into the-AAA protease-mediated dislocation of transmembrane helices in the mitochondrial inner membrane. J. Biol. Chem. 292: 20058-20066. 29030426
Nargund, A.M., C.J. Fiorese, M.W. Pellegrino, P. Deng, and C.M. Haynes. (2015). Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 promotes OXPHOS recovery during the UPR(mt). Mol. Cell 58: 123-133. 25773600
Nargund, A.M., M.W. Pellegrino, C.J. Fiorese, B.M. Baker, and C.M. Haynes. (2012). Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 337: 587-590. 22700657
Pellegrino, M.W., A.M. Nargund, N.V. Kirienko, R. Gillis, C.J. Fiorese, and C.M. Haynes. (2014). Mitochondrial UPR-regulated innate immunity provides resistance to pathogen infection. Nature 516: 414-417. 25274306
Uthoff, M. and U. Baumann. (2018). Conformational flexibility of pore loop-1 gives insights into substrate translocation by the AAA protease FtsH. J Struct Biol 204: 199-206. 30118817