To ensure the removal of excess and non-assembled proteins, mitochondria require a protein quality control system which is constituted by several proteases located in different compartments of mitochondria. m-AAA proteases, oligomeric ATP-dependent metallopeptidases, are key components of this system active at the matrix side of the inner membrane. Human m-AAA proteases build up homo- and hetero-oligomeric complexes composed of AFG3L2 and SPG7. Mice express a third subunit, Afg3l1, resulting in a variety of possible isoenzymes. Interestingly, mutations or deletions of one subunit of mammalian m-AAA proteases cause neurodegeneration in distinct regions of the central and peripheral nervous system in mouse and human, indicating that different tissues, in particular neurons, require a specific subset of isoenzymes. The yeast m-AAA protease can also act as a processing enzyme regulating mitochondrial biogenesis, raising the question which activity is linked to the pathogenesis of the associated diseases. Which molecular functions of mammalian m-AAA proteases contribute to different disease states are poorly understood. Mammalian m AAA proteases have been linked to the processing of the dynamin-like GTPase OPA1 implying a role of mammalian m AAA proteases in mitochondrial fusion. Therefore, to further elucidate the function of mammalian m AAA proteases it was necessary to identify more substrates of the proteases. In this study, a mutation in the Walker B motif of the ATPase domain of Afg3l2/AFG3L2 was identified as dominant negative substrate trap. Using this novel approach, expression of dominant negative mutant variants in human cells, interacting partners and putative substrates have been identified providing further insights into the molecular functions of mammalian m-AAA proteases in mitochondria. These proteases were demonstrated to be present in a supercomplex with prohibitins together regulating cell proliferation and mitochondrial fusion by stabilizing l OPA1. In parallel, m-AAA proteases interact with SLP2 and control stress induced mitochondrial hyperfusion pointing to the formation of another supercomplex containing the proteases and SLP2. The precursor of AFG3L2 itself and MICS1, an inner membrane protein crucial for cristae organization and apoptosis, were identified as possible substrates. Linking m AAA protease functions to mitochondrial fusion, cristae organization and apoptosis may help to unravel the molecular mechanisms underlying neurodegeneration associated with mutations in human m-AAA proteases
