M-AAA and I-AAA Complexes Coordinate to Regulate OMA1, The

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 autocatalytic 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. 1E). Interestingly, we found a significant accumulation of protein to the pro-protein. However, the presence of a residual pre-pro-OMA1 and a corresponding reduction of the pro-OMA1 amount of pro-OMA1 in the absence of the m-AAA complex form. Pre-pro-OMA1 accumulation is not due to alterations in the indicates that an alternative unknown salvage pathway operated by mitochondrial import machinery since we previously demonstrated metalloprotease is active to ensure the generation of the pro-protein. that the absence of AFG3L2 does not affect the import of nuclear Based on our results, we propose that the absence of AFG3L2 encoded proteins into mitochondria (Maltecca et al., 2008). hampers the pre-pro-OMA1 to pro-OMA1 conversion, leading to Importantly, similar results were also obtained with endogenous the accumulation of the pre-pro-protein, but at the same time, the OMA1 (Fig. 1F,G) in both HeLa cells and MEFs. AFG3L2- stress caused by the absence of AFG3L2 (decreased assembling of knockdown HeLa cells showed accumulation of the pre-pro-OMA1 respiratory complexes, ATP depletion, increased ROS production, form and reduction of the pro-OMA1 form (Fig. 1F). Afg3l2−/− accumulation of peptides) activates OMA1 auto-catalytic cleavage, MEFs (Fig. 1G) show a decreased pro-OMA1 form, indicating an leading to a reduction of pro-OMA1. The activated pro-OMA1 is active role of AFG3L2 in pre-pro-OMA1 processing. generated by an autocatalytic processing that involves the final C-terminal amino acids of pro-OMA1 (Baker et al., 2014), causing AFG3L2 physically interacts and processes pre-pro-OMA1 by the removal of the HA moiety from the HA-tagged OMA1 protein. a trimming mechanism In support of this, reintroducing AFG3L2 in Afg3l2−/− cells reduces The diminished conversion of pre-pro-OMA1 into the pro-OMA1 the mitochondrial stress, hence decreasing pro-OMA1 C-terminal form in the absence of AFG3L2 can be explained by a direct auto-cleavage and increasing the amount of pro-OMA1 (Fig. 2A). cleavage of OMA1 by AFG3L2 or by an indirect cleavage mediated by another protease. To discriminate between these alternative YME1L1 regulates pro-OMA1 turnover possibilities, we co-transfected Afg3l2−/− MEFs with OMA1-HA We tested the involvement of YME1L1 in the generation of and AFG3L2-myc or its proteolytic inactive mutant (AFG3L2- pro-OMA1. We failed to demonstrate pre-pro-OMA1 accumulation Q1-myc) and then monitored the processing of both OMA1 and by silencing YME1L1, hence excluding the direct involvement of OPA1. In the absence of AFG3L2, OPA1 is processed to the short YME1L1 in pre-pro-OMA1 processing (Fig. 3A). On the contrary, forms (Ehses et al., 2009; Maltecca et al., 2012) and at the same time depletion of YME1L1 strongly increased the amount of pro-OMA1 pre-pro-OMA1 accumulates (Fig. 2A). (Fig. 3A), confirming a role for YME1L1 in the regulation of The re-expression of AFG3L2 induces a slight increase of OPA1 L1, pro-OMA1 turnover (Rainbolt et al., 2016). so rescuing the physiological processing of OPA1 and inducing an We excluded the increased amount of m-AAA or enhanced increase of pro-OMA1. In contrast, the expression of the proteolytically OMA1 autocatalytic activity (Fig. 3A) or the increased protease inactive form of AFG3L2 cannot restore OMA1 processing (Fig. 2A). activity of the m-AAA complex (Fig. 3B) as possible causes of pro- We therefore explored the possible physical interaction. To stabilize the OMA1 accumulation following YME1L1 knockdown. YME1L1 is interaction, we decided to use AFG3L2-Q1-Myc, which is still capable indeed the specific regulator of pro-OMA1 since in YME1L1- Journal of Cell Science

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Fig. 1. A metallopeptidase cleaves pre-pro-OMA1 to pro-OMA1. (A) Immunoblot analysis of total extracts from HeLa cells transfected with OMA1-HA and treated for 10 h with vehicle (0.001% DMSO), O-phe, E-64 or PMSF. (B) Representative scheme of protease selection. (C) Immunoblot analysis of total extracts from HeLa cells transfected with OMA1-HA, OMA1-Q-HA or empty vector. (D) Immunoblot analysis of total extracts from paraplegin-null immortalized human fibroblasts (HSP) and controls transfected with OMA1-HA or empty vector. (E) Immunoblot analysis of total extracts from Afg3l2 wild type (WT), heterozygous (HET) and knockout (KO) MEFs transfected with OMA1-HA and detected with the indicated antibodies. The graph shows the quantification of the ratio between pre-pro-OMA1 and pro-OMA1 band after OMA1-HA overexpression in MEFs cells by densitometric analysis (ImageJ). (F) Immunoblot analysis of total extracts from HeLa cells transfected with siRNAs against AFG3L2 or OMA1 and treated or not with the uncoupler FCCP for 1 h. The relative graphs show the quantification of the ratio between pre-pro-OMA1 or pro-OMA1 band and Tim44 by densitometric analysis (ImageJ). (G) Immunoblot analysis of total extracts from wild-type, OMA1−/− and AFG3L2−/− MEFs treated or not with the uncoupler FCCP for 1 h. The asterisk indicates the specific endogenous OMA1 band. The graph shows the quantification of the ratio between pro-OMA1 band and Tim44 by densitometric analysis (ImageJ). Bars represent mean±s.d., n=3; *P<0.05, two-tailed Student’s t-test. Journal of Cell Science

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Fig. 2. AFG3L2 is the metallopeptidase converting pre-pro-OMA1 into the pro-OMA1 form. (A) Immunoblot analysis of total extracts from MEFs cells co- transfected with OMA1-HA and empty vector or AFG3L2-myc or AFG3L2-Q1-Myc. (B) Co-immunoprecipitation (Co-IP) of AFG3L2 with OMA1 in HeLa cells after expression of OMA1-HA and AFG3L2-Q1-Myc or empty vector. Protein extracts were immunoprecipitated with anti-myc or anti-HA antibodies. Precipitates were analysed by SDS-PAGE and immunostained using anti-AFG3L2 or anti-HA antibodies. I, input; P, pre-clearing; F, flow through. We immunoprecipitated AFG3L2 and identified OMA1 in the Co-IP lane and the same result was obtained with the reverse approach. (C) Immunoblot analysis of total extracts from

HeLa cells silenced for AFG3L2 and co-transfected with OMA1-HA and empty vector or AFG3L2-ins-myc (AFG3L2-ins-Q1-myc, AFG3L2-ins-Q2-myc). (D) Immunoblot analysis of total extracts from HeLa cells, transfected with OMA1-HA (OMA1 full-length) or OMA1-HA deletion mutants (Δ1-Δ9, see Materials and Methods) as indicated or empty vector. Representative scheme of the mutation sites in OMA1 structure is shown. (E) Immunoblot analysis of total extracts from OMA1-silenced HeLa cells, transfected with OMA1-HA or OMA1-Δ92-144-HA or empty vector. Journal of Cell Science

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Fig. 3. YM1EL1 regulates pro-OMA1 turnover. (A) YME1L1 silencing does not increase the amount of AFG3L2 or OMA1 autocatalytic activity. Immunoblot analysis of total extracts from HeLa cells silenced for AFG3L2, YME1L1 or both transfected with OMA1-HA or empty vector. The graph shows quantification of pro-OMA1 after OMA1-HA overexpression in HeLa cells silenced for AFG3L2, YME1L1 or both by densitometric analysis (ImageJ). Bars represent mean±s.d., n=3; *P<0.05, two-tailed Student’s t-test. (B) YME1L1 silencing does not increase AFG3L2 catalytic activity. AFG3L2 specifically cleaves precursor MrpL32 to the mature form and the absence of AFG3L2 causes accumulation of unprocessed MrpL32 (Nolden et al., 2005), whereas the absence of YME1L1 has no effect on MrpL32 maturation. Immunoblot analysis of total extracts from HeLa cells silenced for AFG3L2, YME1L1 or for both, transfected with MrpL32-HA or empty vector. (C) Pro-OMA1 increases upon YME1L1 depletion. Immunoblot analysis of total extracts from HeLa cells silenced for YME1L1 and co-transfected with OMA1-HA and YME1L1-ins-myc or YME1L1-ins-Q-myc or empty vector and detected with the indicated antibodies. knockdown cells pro-OMA1 accumulates (Fig. 3C). Therefore, We showed that AFG3L2 is crucial for OMA1 maturation, hence YME1L1 is directly involved in regulating the pro-OMA1 level, directly allowing its conversion from pre-pro-OMA1 to pro-OMA1 thus preventing OMA1 accumulation, OPA1 over-processing and by a trimming mechanism involving the solvent-exposed the consequent mitochondrial network fragmentation. N-terminal part of the protein. Possibly, another as yet unknown Taken together, these data support for the first time that the protease could be involved in the generation of pro-OMA1, m-AAA and i-AAA complexes actively cooperate in controlling highlighting the existence of a compensatory mechanism acting mitochondrial dynamics through the fine regulation of OMA1 when AFG3L2 is absent or mutated. We indeed demonstrated that processing and stability. The vital necessity of accurately the i-AAA complex is involved in the regulation of pro-OMA1 controlling OMA1 proteolytic activity is underlined by the turnover, preventing its accumulation and, consequently, findings of a ‘tipping-point’ threshold of Δϕ due to both DRP1 mitochondrial network fragmentation. and OMA1, which act coordinately to balance the fusion–fission A possible model of functional interactions between m-AAA, equilibrium under Δϕ stimulus (Jones et al., 2017). i-AAA and OMA1 is shown in Fig. 4. Under physiological Journal of Cell Science

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Fig. 4. m-AAA and i-AAA complexes regulate OMA1 processing. Model of OMA1 processing and regulation. (A) In physiological conditions, pre-pro-OMA1 interacts with AFG3L2 after mitochondrial import. This generates pro-OMA1, which is regulated by the activity of the i-AAA, thus avoiding its accumulation. In these conditions, OMA1 and YME1L1 perform their constitutive cleavage on OPA1 (at S1 and S2 sites, respectively) ensuring the balance between OPA1 long and short forms and hence a physiological mitochondrial network. The yellow rectangle of OMA1 transmembrane domain represents the leucine stretch halting the m-AAA trimming. (B) In the absence of AFG3L2 (m-AAA), the efficiency of pre-pro-OMA1 to pro-OMA1 conversion is minimized. This induces several stress events that are able to activate OMA1. (C) In the absence of YME1L1, pro-OMA1 accumulates, which, together with the stress caused by the absence of the i-AAA complex, induces OMA1 activation. In both B and C, the OMA1 increment enhances its proteolytic activity towards OPA1, thus causing mitochondrial network fragmentation. conditions, AFG3L2 cleaves pre-pro-OMA1 to generate abundant proteostatic stresses (Maltecca et al., 2008; Richter et al., 2015) that pro-OMA1, which is further controlled by the activity of i-AAA, possibly turn on an emergency pathway operated by other proteases which regulates pro-OMA1 accumulation and ensures a balance that activate OMA1 and the downstream effect of mitochondrial between long and short OPA1 forms. fragmentation. The same effect is caused by the absence of YME1L1 In contrast, the absence of AFG3L2 induces a wide range of and the resultant pro-OMA1 accumulation, which enhances organellar damage, including defective assembly of respiratory its autocatalytic activity and, again, induces mitochondrial complexes, ROS production, ATP depletion and inner membrane fragmentation. Considering the critical role of OMA1 in the Journal of Cell Science

6 SHORT REPORT Journal of Cell Science (2018) 131, jcs213546. doi:10.1242/jcs.213546 mitochondrial fusion–fission homeostasis, it is conceivable that antibody and anti-GAPDH were from Santa Cruz Biotechnology (sc-32239, multiple alternative effectors, including AFG3L2, YME1L1 and 1:1000 and sc-32233, 1:10,000), anti-c-Myc and anti-OMA1 were from OMA1 itself, together with Δϕ, are needed to maintain the effective Novus Biologicals (Littleton, CO, USA, NB600-335, 1:1000 and NBP2- fine-tuning of this crucial stress sensor protein. 30971, 1:500), anti-YME1L1 antibody was from ProteinTech Group (Chicago, IL, USA, 11510-AP, 1:1000), anti-Hsp60 was from Enzo Life Science (Farmingdale, NY, USA, ADI-SPA-806, 1:7000) and anti-AFG3L2 MATERIALS AND METHODS was previously generated in the lab (Atorino et al., 2003) and used at 1:2000. Transfection constructs For the generation of pcDNA3.1-Oma1-HA (herein OMA1-HA), Oma1 mouse cDNA clone was obtained from Thermo Scientific (Waltham, MA, Protease inhibitors USA) and an HA tag sequence introduced. The proteolytically inactive HeLa cells were transfected with OMA1-HA for 14 h and subsequently OMA1E324Q-HA was obtained by site-specific mutagenesis of pcDNA3.1- incubated for 10 h with the following protease inhibitors: 0.5 mM O-phe, 100 µM E-64d and 400 µM PMSF (Sigma-Aldrich, St Louis, MO, USA). Oma1-HA. To generate pcDNA3.1-deleted-Oma1-HA mutants (in the text: Δ1-Δ9) we introduced the indicated deletions performing site-directed mutagenesis: Δ1, pcDNA3.1-Oma1-Δ147-149-HA; Δ2, pcDNA3.1-Oma1- Co-immunoprecipitation Δ150-152-HA; Δ3, pcDNA3.1-Oma1-Δ153-155-HA; Δ4, pcDNA3.1-Oma1- One mg protein was extracted from HeLa cells in lysis buffer (50 mM Tris- Δ169-172-HA; Δ5, pcDNA3.1-Oma1-Δ170-HA; Δ6, pcDNA3.1-Oma1- HCl, pH 8, 150 mM NaCl, 0.5 mM EDTA pH 8, 1× protease inhibitor Δ171-HA; Δ7, pcDNA3.1-Oma1-Δ172-HA; Δ8, pcDNA3.1-Oma1-Δ175- cocktail, 0.15% Sarkosyl), after mild sonication. After a pre-clearing step – 178-HA; Δ9, pcDNA3.1-Oma1-Δ177-180-HA. (incubation with Protein-G Sepharose beads) proteins were incubated with In pcDNA3.1-Oma1-Δ92-144-HA (herein OMA1-Δ92-144-HA), we anti-Myc or anti-HA (Clone 16B12 monoclonal antibody, Affinity Matrix introduced a deletion between amino acids 92 and 144. beads, Covance, Princeton, NJ, USA) antibodies for 1 h with constant gentle – pcDNA3.1-AFG3L2-Myc (AFG3L2-myc), was previously described agitation. Subsequently, Protein-G Sepharose was added. Precipitates were (Maltecca et al., 2012). pcDNA3.1-AFG3L2-E408Q-Myc and pcDNA3.1- used for western blot analysis. AFG3L2-E575Q-Myc appear in the text as AFG3L2-Q1-Myc and AFG3L2- Q2-Myc, respectively. Uncoupler treatment In order to make them insensitive to the stealth RNAi against AFG3L2, HeLa cells were treated with 10 µM carbonyl cyanide-4- these constructs were mutagenized without changing the sense of encoded (trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich) for 1 h. amino acid sequence, generating: pcDNA3.1-AFG3L2-Ins-Myc, pcDNA3.1- AFG3L2-Ins-E575Q-Myc (AFG3L2-Ins-Q1-Myc), pcDNA3.1-AFG3L2-Ins- Acknowledgements We are grateful to Laura Cassina for scientific discussion; Maurizio De Fusco for E408Q-Myc (AFG3L2-Ins-Q2-Myc). pMT21-YME1L1-Myc (YME1L1-Myc) was previously described technical assistance; Michela Riba and Davide Gaudesi for bioinformatic analyses. OMA1−/− MEF cells were kindly provided by Carlos Lopez-Otin, Universidad de (Coppola et al., 2000). pMT21-YME1L1-E543Q-Myc (YME1L1-Q-Myc) Oviedo, Oviedo, Spain. was generated by site-directed mutagenesis. To make them insensitive to the stealth RNAi against YME1L1, these constructs were mutagenized without Competing interests changing the sense of encoded amino acid sequence. MrpL32 cDNA was The authors declare no competing or financial interests. isolated from HeLa cells, HA-tagged and cloned into pCDNA3.1 (MRPL32-HA in the text). All primers are available on request. We Author contributions confirmed all constructs by Sanger sequencing. Conceptualization: F.M., G.C.; Methodology: F.C., S.T., I.S.; Investigation: F.C., F.M., S.T.; Writing - original draft: F.C., F.M., I.S., G.C.; Writing - review & editing: Cell culture G.C.; Funding acquisition: G.C. Human immortalized fibroblasts, MEFs and HeLa cells were maintained in DMEM containing penicillin-streptomycin 10 μg/ml, 10% fetal bovine serum, Funding 2 mM L-glutamine and 1 mM sodium pyruvate. Transient transfections for the This work was supported by the Italian Telethon Foundation (GGP12235), Fondazione Cariplo (2012_0646), Ministero Italiano dell’Universitàe della Ricerca overexpression of the indicated constructs or for the silencing of the indicated (MIUR, 20108WT59Y_001), Associazione Italiana Sindromi Atassiche (AISA), the genes were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, National Ataxia Foundation (NAF) and the Ministero Italiano della Salute (Giovani ’ USA) according to the manufacturer s instructions. Ricercatori GR-2011-02351638 to F.M.). mRNA silencing References Knockdown of AF3GL2, OMA1 and YME1L1 was performed using Anand, R., Wai, T., Baker, M. J., Kladt, N., Schauss, A. C., Rugarli, E. and specific stealth RNAi siRNAs and scrambled stealth RNAi siRNAs as a Langer, T. (2014). The i-AAA protease YME1L and OMA1 cleave OPA1 to negative control (Invitrogen, Carlsbad, CA, USA). The sequences of the balance mitochondrial fusion and fission. J. Cell Biol. 204, 919-929. Ashburner, M., Ball, C. A., Blake, J. A., Botstein, D., Butler, H., Cherry, J. M., stealth RNAi siRNAs are 5′-acgacuuccaaucugucuacuacuc-3′ for human ′ ′ ′ Davis, A. P., Dolinski, K., Dwight, S. S., Eppig, J. T. et al. (2000). Gene AFG3L2,5-uggacuacugcuugcugcaaaggcu-3 for human OMA1,5-ucca- ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. ′ gaaacccaaucugccaucgaa-3 for human YME1L1; 100 pmoles of each siRNA Genet. 25, 25-29. were transfected. Down-regulation of the target genes was monitored by Atorino, L., Silvestri, L., Koppen, M., Cassina, L., Ballabio, A., Marconi, R., immunoblot analysis on cell lysates 72 h after transfection. Langer, T. and Casari, G. (2003). Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia. J. Cell Biol. 163, 777-787. Western blot analysis Baker, M. J., Lampe, P. A., Stojanovski, D., Korwitz, A., Anand, R., Tatsuta, T. and Western blot was performed using standard protocols. Briefly, total cell Langer, T. (2014). Stress-induced OMA1 activation and autocatalytic turnover lysate was prepared by protein extraction using the following buffer: 50 mM regulate OPA1-dependent mitochondrial dynamics. EMBO J. 33, 578-593. Tris-HCl, pH 8, 150 mM NaCl, 2% Triton X-100, 0.5 mM EDTA, pH 8, 1× Bonn, F., Tatsuta, T., Petrungaro, C., Riemer, J. and Langer, T. (2011). protease inhibitor cocktail (Roche Applied Science, Penzberg, Upper Presequence-dependent folding ensures MrpL32 processing by the m-AAA Bavaria, Germany). 25 µg of protein extracts were dissolved in sample protease in mitochondria. EMBO J. 30, 2545-2556. buffer (60 mM Tris-HCl, pH 6.8, 5% glycerol, 1.7% SDS, 0.1 M DTT, Cipolat, S., Martins de Brito, O., Dal Zilio, B. and Scorrano, L. (2004). OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc. Natl. Acad. Sci. USA 0.002% Bromophenol Blue), were resolved by SDS-PAGE and analysed 101, 15927-15932. by standard immunoblotting procedures. Anti-HA antibody was from Coppola, M., Pizzigoni, A., Banfi, S., Bassi, M. T., Casari, G. and Incerti, B. Sigma-Aldrich (H6908, 1:2000), anti-OPA1 was from BD Transduction (2000). Identification and characterization of YME1L1, a novel paraplegin-related

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