© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs213546. doi:10.1242/jcs.213546 SHORT REPORT m-AAA and i-AAA complexes coordinate to regulate OMA1, the stress-activated supervisor of mitochondrial dynamics Francesco Consolato1,*, Francesca Maltecca1,*, Susanna Tulli1, Irene Sambri2 and Giorgio Casari1,2,‡ ABSTRACT and fission (L and S forms, respectively) (Anand et al., 2014). The The proteolytic processing of dynamin-like GTPase OPA1, mediated balance between long and short OPA1 forms is finely regulated by two by the activity of both YME1L1 [intermembrane (i)-AAA protease mitochondrial inner membrane proteases, OMA1 (Ehses et al., 2009) complex] and OMA1, is a crucial step in the regulation of mitochondrial and YME1L1, which cleave OPA1 at different sites (Song et al., 2007). dynamics. OMA1 is a zinc metallopeptidase of the inner mitochondrial The intermembrane (i)-AAA protease YME1L1 exposes its membrane that undergoes pre-activating proteolytic and auto- catalytic domain to the intermembrane space (Leonhard et al., 1996) proteolytic cleavage after mitochondrial import. Here, we identify and is responsible for generation of the S2-OPA1 form by proteolytic AFG3L2 [matrix (m)-AAA complex] as the major protease mediating cleavage, whereas OMA1 gives rise to the S1 and S3 forms (Anand this event, which acts by maturing the 60 kDa pre-pro-OMA1 to the et al., 2014; MacVicar and Langer, 2016; Quirós et al., 2012). 40 kDa pro-OMA1 form by severing the N-terminal portion without OMA1 harbours an M48 metallopeptidase domain and is the major recognizing a specific consensus sequence. Therefore, m-AAA and player in OPA1 processing under conditions of stress (Quirós et al., Δϕ i-AAA complexes coordinately regulate OMA1 processing and 2012). Different stress stimuli, such as dissipation of , increased turnover, and consequently control which OPA1 isoforms are ROS production, decreased ATP level, heat shock and loss of present, thus adding new information on the molecular mechanisms mtDNA have been demonstrated to overactivate OMA1 (Anand of mitochondrial dynamics and neurodegenerative diseases affected et al., 2014; Baker et al., 2014; Ehses et al., 2009; Head et al., 2009). Δϕ by these phenomena. Recently, a threshold of was identified as a determinant of mitochondrial homeostasis mediated by OMA1 and DRP1, which This article has an associated First Person interview with the first cooperatively regulate OPA1 maintenance and processing, and author of the paper. therefore control fission and fusion pathways (Jones et al., 2017). The absence of YME1L1, which generates organellar stress, also KEY WORDS: OMA1, OPA1, m-AAA complex, Mitochondrial activates OMA1 (Stiburek et al., 2012); in fact, YME1L1 and dynamics OMA1 are reciprocally regulated in response to specific stress stimuli. OMA1 is degraded by YME1L after toxic insults that INTRODUCTION depolarize the mitochondrial membrane and cause ATP depletion. The mitochondrial network optimizes different activities by The balance of degradation activities of these two proteins tunes continuously changing its morphology by the essential and the proteolytic processing of OPA1, which, in turn, affects the antagonistic activities of fission and fusion (Pernas and Scorrano, mitochondrial morphology state, thus profoundly modulating 2016; Westermann, 2010). These dynamic processes are regulated mitochondrial functions (Rainbolt et al., 2016). by the pro-fission dynamin-related proteins (DRPs), DRP1, Fis1 OMA1 is synthesized as a pre-pro-protein of 60 kDa that and MFF1 (Gandre-Babbe and van der Bliek, 2008; James et al., undergoes proteolytic processing upon import into mitochondria 2003; Smirnova et al., 2001), and pro-fusion proteins, MFN1, to generate a mature form of 40 kDa (Baker et al., 2014; Head et al., MFN2 and OPA1, which are involved in the fusion of the outer and 2009). It has been proposed that 40 kDa OMA1 is a stress-sensitive inner mitochondrial membrane (OMM and IMM), respectively pro-protein that undergoes autocatalytic cleavage of a C-terminal (Cipolat et al., 2004; Pernas and Scorrano, 2016). subunit peptide upon stress stimuli to generate the active form of The GTPase protein OPA1 exists in different splicing isoforms that OMA1 that is ultimately responsible for OPA1 processing (Baker can be partially or totally proteolytically processed at one or two et al., 2014). All these findings indicate that OMA1 is a key sensor distinct sites (S1 and S2) (Delettre et al., 2001). The combination for a plethora of mitochondrial stress events. of splicing isoforms and proteolytic cleavage results in five Nevertheless, clear information on the generation of pro-OMA1 electrophoretically distinguishable protein bands, named L1, L2 (the and the proteases involved in this mechanism is still missing. Here, long forms) and S1, S2 and S3 (the short forms), which promote fusion we add an additional tile to this complex system of interlaced and regulated proteases by showing that (1) AFG3L2, the essential component of the matrix (m)-AAA complex, processes OMA1 by 1Vita-Salute San Raffaele University and Neurogenomics Unit, Division of Genetics and Cell Biology, San Raffaele Scientific Institute, 20132 Milano MI, Italy. 2Genomic mediating its conversion from pre-pro-OMA1 to pro-OMA1, Medicine Program, Telethon Institute of Genetics and Medicine (TIGEM), 80078 whereas (2) YME1L1 is tightly involved in pro-OMA1 catabolism, Pozzuoli NA, Italy. as previously suggested (Rainbolt et al., 2016). *These authors contributed equally to this work ‡Author for correspondence ([email protected]) RESULTS AND DISCUSSION AFG3L2 mediates OMA1 initial processing F.C., 0000-0002-2229-2644; F.M., 0000-0001-8095-8208; S.T., 0000-0001- 5504-4121; I.S., 0000-0003-3500-6958; G.C., 0000-0002-0115-8980 With the aim of identifying the peptidase involved in OMA1 processing, HeLa cells were transfected with OMA1-HA, and Received 24 November 2017; Accepted 13 March 2018 inhibitors of different classes of peptidases were added to monitor Journal of Cell Science 1 SHORT REPORT Journal of Cell Science (2018) 131, jcs213546. doi:10.1242/jcs.213546 the accumulation of the pre-pro-OMA1 form: PMSF to inhibit of substrate binding but lacks proteolytic activity (Atorino et al., 2003), serine proteases, E64 for cysteine proteases and O-phe to block as a trap for interacting proteins. As hypothesized, AFG3L2 co- metallopeptidases (see the Materials and Methods). Only O-phe immunoprecipitates with OMA1 (Fig. 2B). treatment induced the accumulation of the pre-pro-OMA1 form, To gain further evidence of the role of AFG3L2 in OMA1 indicating that the class of metallopeptidase is the one involved in processing in human cells, which unlike murine cells do not express the initial processing of the protein (Fig. 1A). the AFG3L2 homologue AFG3L1, we tested the accumulation A total of 380 putative mitochondrial proteases were selected from of pre-pro-OMA1 in HeLa cells silenced for AFG3L2 and four different protein databases: UniProt (UniProt Consortium, 2013), demonstrated that they behave similarly to Afg3l2−/− MEFs. In MitoCarta (Pagliarini et al., 2008), MitoMiner (Smith and Robinson, fact, upon silencing of AFG3L2, we detected the accumulation of 2009) and Gene Ontology (Ashburner et al., 2000). Combining O-phe the pre-pro-OMA1 and the presence of intermediate bands between sensitivity with the presence of the protease in at least two of the pre-pro-OMA1 and pro-OMA1 (Fig. 2C, first two lanes). Moreover, databases, we reduced candidates to 27 proteins. Among them, all expression of proteolytic siRNA-insensitive AFG3L2 mutants proteins inhibited by metal ions or recognizing specific consensus (AFG3L2-ins-Q1-Myc, AFG3L2-ins-Q2-Myc) failed to reduce the sequence not present in OMA1 or showing deacetylase/hydrolase amount of the pre-pro-OMA1 form (Fig. 2C, right panel). activity were excluded. Since OMA1 is localized in the IMM (Baker In order to identify the putative target sequence, a series of et al., 2014) and presumably its cleavage occurs in this site, four segmentally deleted OMA1 mutants revealed that pre-pro-OMA1 to proteins localized in the IMM were prioritized: paraplegin (encoded pro-OMA1 maturation is the consequence of an N-terminal trimming by the SPG7 gene), AFG3L2, YME1L1 and OMA1 (Fig. 1B). effect operated by the m-AAA complex, similar to maturation of We excluded OMA1 itself from this initial step since the expression MRPL32, another m-AAA-specific substrate (Bonn et al., 2011) of the proteolytic inactive mutant (OMA1-Q-HA) does not affect the (Fig. 2D). Moreover, since the N-terminus of 40 kDa OMA1 has been pre-pro OMA1 to pro-OMA1 cleavage (Fig. 1C). However, we mapped at amino acid 140 (Baker et al., 2014), we generated a vector confirmed that OMA1 has an active role on its own processing, in expressing a truncated form of OMA1 that mimics pro-OMA1 by particular in the conversion from the pro-OMA1 to the fragment of removing 53 amino acids between the mitochondrial leader sequence ∼30 kDa, as previously reported (Baker et al., 2014). Indeed, we and the first N-terminal amino acids of pro-OMA1. This mutant observed that the expression of OMA1-Q-HA prevents the processing (OMA1-Δ92-144-HA) encodes a protein of 50 kDa and is processed to the 30 kDa OMA1 fragment, demonstrating that it is generated by like the wild-type protein to generate pro-OMA1 (Fig. 2E). We also auto-processing (Fig. 1C). By transfecting OMA1-HA in HSP patient found that OMA1-Δ92-144 is proteolytically active since it is able to fibroblasts lacking paraplegin (Atorino et al., 2003), we found no cleave L-OPA1, restoring the bands S1 and S3, which both alteration of OMA1 processing (Fig. 1D), thus excluding paraplegin disappeared after OMA1 silencing (Fig. 2E). from candidates and confirming previous findings (Ehses et al., 2009). Our findings demonstrate the major role of AFG3L2 in OMA1 We then evaluated OMA1 processing in the absence of AFG3L2 in processing, and in particular, in the conversion from the pre-pro- MEFs (Fig.