The Plant I-AAA Protease Controls the Turnover of an Essential Mitochondrial Protein Import Component Magdalena Opalińska1,‡, Katarzyna Parys1,*, Monika W

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SHORT REPORT SPECIAL ISSUE: PLANT CELL BIOLOGY The plant i-AAA protease controls the turnover of an essential mitochondrial protein import component Magdalena Opalińska1,‡, Katarzyna Parys1,*, Monika W. Murcha2,3 and Hanna Jańska1,‡

ABSTRACT ATP-dependent proteases with versatile activities are engaged in the Mitochondria are multifunctional organelles that play a central role maintenance of mitochondrial protein homeostasis (proteostasis) in energy metabolism. Owing to the life-essential functions of and function (Voos, 2013; Quirós et al., 2015; Janska et al., 2010). these organelles, mitochondrial content, quality and dynamics are The mitochondrial inner membrane-embedded AAA protease tightly controlled. Across the species, highly conserved ATP-dependent (denoted i-AAA) forms a homo-oligomeric ATP-dependent proteases prevent malfunction of mitochondria through versatile proteolytic complex that is embedded in the inner mitochondrial activities. This study focuses on a molecular function of the plant membrane (IMM) with the catalytic sites exposed to the mitochondrial inner membrane-embedded AAA protease (denoted i- intermembrane space (IMS) (Gerdes et al., 2012). In humans, a AAA) FTSH4, providing its first bona fide substrate. Here, we report that homozygous mutation in the gene encoding i-AAA protease the abundance of the Tim17-2 protein, an essential component of the (YME1L) was found to be associated with the development of TIM17:23 translocase (Tim17-2 together with Tim50 and Tim23), is mitochondriopathy with optic nerve atrophy (Hartmann et al., directly controlled by the proteolytic activity of FTSH4. Plants that are 2016). Upon loss or depletion of i-AAA protease, both in yeast and lacking functional FTSH4 protease are characterized by significantly in mammals, pleiotropic phenotypes are observed. Yeast and enhanced capacity of preprotein import through the TIM17:23- mammalian i-AAA proteases display diverse activities, including dependent pathway. Taken together, with the observation that FTSH4 proteolytic processing of mitophagy and fusion factors, selective prevents accumulation of Tim17-2, our data point towards the role of this elimination of misfolded and unassembled subunits, and control of i-AAA protease in the regulation of mitochondrial biogenesis in plants. the turnover of specific regulatory proteins that altogether influence a wide spectrum of processes occurring at the mitochondria KEY WORDS: AAA protease, ATP-dependent proteolysis, (Thorsness et al., 1993; Weber et al., 1996; Nebauer et al., 2007; Mitochondrial protein import, TIM17:23 translocase Ruan et al., 2013; Stiburek et al., 2012; Anand et al., 2014; Wang et al., 2013; Potting et al., 2010; Nakai et al., 1995). INTRODUCTION Plant mitochondria contain two i-AAA proteases, FTSH4 and Mitochondria are essential, double membrane-bound organelles that FTSH11 (Urantowka et al., 2005). Arabidopsis thaliana devoid of are the main sites of ATP production in eukaryotic cells. Continuous FTSH4 displays severe developmental and morphological changes degradation and biogenesis processes enable the maintenance of under temperature-stress conditions (Dolzblasz et al., 2016; healthy mitochondrial populations and allow the mitochondrial Smakowska et al., 2016). These alterations correlate with proteome to adapt in response to the fluctuating cellular oxidative stress, imbalance in phospholipid metabolism, requirements (Nunnari and Suomalainen, 2012; Harbauer et al., perturbation in respiratory chain complexes activity and aberrant 2014; Palikaras and Tavernarakis, 2014). The vast majority of mitochondrial morphology (Smakowska et al., 2016). Similarly, mitochondrial proteins are nuclear encoded and synthetized as loss of FTSH4 affects development and leaf morphology at the late preproteins in the cytosol that are transported into destined sub- stage of rosette growth when subjected to a short-day photoperiod at mitochondrial localization through the action of sophisticated, optimum temperature (Gibala et al., 2009; Kolodziejczak et al., membrane-embedded protein translocation machineries 2007). In contrast to yeast or mammalian systems, the endogenous (Wasilewski et al., 2016; Schulz et al., 2015). substrates of i-AAA protease have not yet been described in plants Mitochondrial integrity considerably depends on balancing the and consequently, the molecular mechanisms detailing how FTSH4 levels of different subunits of protein complexes. This is achieved maintains mitochondrial function remain elusive. through various mechanisms, including selective removal of excess Here, we demonstrate that plant i-AAA protease FTSH4 is subunits by ATP-dependent proteolysis (König et al., 2016; required for the turnover of the essential mitochondrial inner Szczepanowska et al., 2016). Within the mitochondria, diverse membrane preprotein translocase Tim17-2. The lack of functional FTSH4 protease is associated with an increased abundance of 1Faculty of Biotechnology, University of Wroclaw, F. Joliot-Curie 14A, Wroclaw 50- Tim17-2 and an enhanced import capacity of model substrates for the 383, Poland. 2Australian Research Council Centre of Excellence in Plant Energy TIM17:23 translocase; Tim17-2 together with Tim50 and Tim23 Biology, University of Western Australia, 35 Stirling Highway, Crawley Western Australia 6009, Australia. 3School of Molecular Sciences, University of Western form the core of the TIM17:23 complex (Wang et al. 2012). This Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia. study provides the first identification of the physiological substrate of *Present address: Gregor Mendel Institute, Austrian Academy of Sciences, Vienna plant i-AAA protease and links FTSH4 protease and mitochondrial Biocenter, A-1030 Vienna, Austria. preprotein import machinery. ‡Authors for correspondence ([email protected]; [email protected]) RESULTS AND DISCUSSION M.O., 0000-0003-1449-4243 Mitochondria devoid of FTSH4 accumulate Tim17-2 To understand the role of FTSH4 protease in the maintenance of

Received 15 December 2016; Accepted 1 March 2017 mitochondrial function, we searched for primary changes caused by Journal of Cell Science

1 SHORT REPORT Journal of Cell Science (2018) 131, jcs200733. doi:10.1242/jcs.200733 the loss of this i-AAA protease. Consequently, to diminish the risk we found a substantial increase in the steady-state levels of Tim17-2 of secondary effects, we sought to identify mitochondrial proteins in mitochondria isolated from both ftsh4-1 and ftsh4-2 lines (Fig. 1; that accumulate in ftsh4 mutants cultivated under optimal growth Fig. S1). Tim17-2 is a highly conserved, essential component of the conditions, where no significant alterations in mitochondrial inner mitochondrial membrane protein translocase – the TIM17:23 activity were observed (22°C with a long-day photoperiod, cycles complex – that transports the majority of mitochondrial proteome of 8 h dark and 16 h light) (Smakowska et al., 2016). Interestingly, (Wasilewski et al., 2016; Schulz et al., 2015). As evident from studies performed in yeast, Tim17 plays multiple roles within the TIM17:23 translocase. It regulates pore structure and voltage gating of the Tim23 channel, and is required for the promotion of the inner- membrane insertion of preproteins and direct interaction of the TIM17:23 core with the presequence translocase-associated motor (Martinez-Caballero et al., 2007; Chacinská et al., 2005). Consequently, the precise level and stoichiometry of Tim17 may be important for the optimal functioning of TIM17:23. A. thaliana contains three isoforms of Tim17, of which only Tim17-2 is essential and constitutively expressed (Murcha et al., 2014). We found that steady-state levels of other TIM17:23 subunits as well as levels of components of protein translocases from the outer mitochondrial membrane, Sam50 and Tom40, and of the intermembrane space-localized small Tim chaperone, Tim9, were unaffected upon FTSH4 loss (Fig. 1). These results show that the increase in Tim17-2 levels in ftsh4 mutants is specific and not caused by a general upregulation of mitochondrial preprotein import machineries. To further address correlation between the FTSH4

Fig. 2. A substantial portion of Tim17-2 is present as low molecular mass sub-complexes in mitochondria lacking FTSH4 protease. (A) Western blot analysis of mitochondrial fractions from wild type and ftsh4 mutants subjected to alkaline carbonate extraction. Equal amounts from the whole mitochondria (Total), supernatant fractions (soluble and peripheral membrane Fig. 1. Tim17-2 protein levels are elevated in ftsh4 mutant. Representative proteins) and pellet fractions (membrane-integrated proteins) were resolved immunoblots of mitochondrial fractions prepared from wild type and ftsh4-1 with SDS-PAGE, immobilized on PVDF membrane and analyzed with specific cells (upper panel). Quantification of steady-state levels of mitochondrial antibodies. (B) Mitochondria isolated from wild-type and ftsh4-1 plants were proteins in ftsh4 mutant relatively to wild type (set to 1). Bars represent lysed with digitonin and subjected to BN-PAGE followed by SDS-PAGE in the mean±s.d. from n≥3 (lower panel). IM, inner membrane; OM, outer membrane; second dimension. TIM17:23 complexes were probed with anti-Tim17-2 and

IMS, intermembrane space. -Tim50 antibodies. Journal of Cell Science

2 SHORT REPORT Journal of Cell Science (2018) 131, jcs200733. doi:10.1242/jcs.200733 protease and Tim17-2 levels we generated a ftsh4-1 (Wang et al., 2012). A small portion of Tim17-2 in a low molecular complementation line ( ftsh4-1 FTSH4), in which FTSH4 was mass sub-complex was detected. In contrast, a substantial fraction of expressed under the constitutive CaMV 35S promotor (Fig. S2). Tim17-2 was present as a low molecular mass sub-complex in ftsh4 Overproduction of FTSH4 rescued the morphological phenotype of mutant. Furthermore, the ratio of TIM17:23 complexes in ftsh4 ftsh4-1 line (Fig. S3) and led to a decrease in Tim17-2 steady-state mutants was noticeably altered. Taken together, we conclude that levels (Fig. S2). FTSH4 controls the abundance of Tim17-2 in the mitochondrial Next, we questioned whether Tim17-2 present in excessive membrane and that lack of this protease exerts an impact on the amounts in ftsh4 mutants is correctly integrated into the architecture of TIM17:23 translocase. mitochondrial membrane. To address this, we isolated mitochondria from wild-type and mutant plants and performed alkaline carbonate Proteolytic activity of FTSH4 is required to prevent extraction that allows for distinguishing between integral membrane accumulation of Tim17-2 proteins (pellet) and soluble or peripheral membrane proteins Specific accumulation of Tim17-2 in ftsh4 mutant mitochondria (supernatant). Both in wild-type and ftsh4 mitochondria, Tim17-2 strongly suggests that FTSH4 proteolytic activity is required for the was exclusively found in pellet fractions after sodium carbonate turnover of this essential protein import component. In order to treatment, indicating integration into the inner membrane (Fig. 2A). address whether Tim17-2 represents a proteolytic substrate of To confirm specificity of the assay, mtHsp70 (also known as FTSH4, we generated a mutant line expressing a proteolytically mtHsc70-1, a soluble protein of mitochondrial matrix) and Slp1 inactive variant of FTSH4 [FTSH4(H486Y)] in the ftsh4-1 (mitochondrial inner membrane protein that is partially extracted with background. The overproduction of FTSH4(H486Y) was sodium carbonate; Gehl et al., 2014) were used as controls. confirmed by immunoblot analysis (Fig. 3A). Expression of We also addressed Tim17-2 distribution within mitochondrial the proteolytically inactive variant of FTSH4 neither restored membrane protein complexes by Blue native (BN)-PAGE followed the morphological phenotype of ftsh4-1 line (Fig. S3) nor by a second-dimension electrophoresis under denaturing conditions downregulated levels of Tim17-2 (Fig. 3A), demonstrating the (2D-BN/SDS-PAGE) (Fig. 2B). In wild-type mitochondria, Tim17- importance of the proteolytic function of this i-AAA protease 2 mainly co-migrated with Tim50 as does the TIM17:23 translocase in vivo. To obtain further evidence for the role of FTSH4 proteolytic

Fig. 3. Proteolytic activity of FTSH4 is required to prevent Tim17-2 accumulation. (A) Representative immunoblot of mitochondrial fractions prepared from wild-type, ftsh4-1 and ftsh4-1 FTSH4(H486Y) plants (left panel). The density of the Tim17-2 band is shown underneath the blot (Qunatif.). Quantification of steady- state levels of Tim17-2 and SHMT in ftsh4-1 FTSH4 (H486Y) mutants relative to wild type (set to 1). Bars represent mean±s.d. from n>3 (right panel). (B) Immunoblot of purified mitochondria obtained from ftsh4-1 FTSH4(H486Y) and wild-type plants. Mitochondria were incubated for 0 to 4 h at 35°C in a buffer supplemented with ATP (upper panel). Quantification of Tim17-2 and Slp1 stability in ftsh4-1 FTSH4(H486Y) mutant and wild-type mitochondria. Results represent mean±s.d. from n=3 (lower panel). (C) Immunoblot showing co-immunoprecipitation of Tim17-2 with FLAG-tagged proteolytically inactive FTSH4. Mitochondria from ftsh4-1 FTSH4(H486Y) and ftsh4-1 control were lysed with digitonin and immunoprecipitated with anti-FLAG affinity matrix. The precipitated proteins were then immunoblotted with antibodies against the indicated proteins. IN, input (5%); FT, flow-through (5%); W, wash; E, eluate. (D) Immunoblot showing co-immunoprecipitation of Tim17-2 with FLAG-tagged proteolytically inactive FTSH4. Mitochondria from ftsh4-1 FTSH4(H486Y) were lysed with digitonin and incubated with or without antibodies raised against Tim17-2 and Protein-A– Sepharose. The precipitated proteins were then immunoblotted with antibodies against the indicated proteins. W, wash; E, eluate. Journal of Cell Science

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Fig. 4. Lack of FTSH4 protease promotes mitochondrial protein import through the TIM17:23-dependent pathway. (A) Radiolabeled preprotein AOX was incubated at 26°C with isolated wild-type and ftsh4-1 mitochondria for the indicated times. After protease K treatment, the samples were resolved by SDS-PAGE and analyzed by digital autoradiography. The amount of the processed AOX in wild-type mitochondria after the longest import time was set to 100% (control). Results represent mean±s.e.m. from n>3. (B) Radiolabeled carrier preprotein ANT was incubated at 26°C with isolated wild-type and ftsh4-1 mitochondria for the indicated times. After protease K treatment, the samples were resolved by SDS-PAGE and analyzed by digital autoradiograpy. The amount of the processed ANT in wild-type mitochondria after the longest import time was set to 100% (control). Results represent mean±s.e.m. from n=3. p, precursor; m, mature. activity in the regulation of Tim17-2 levels, we analyzed turnover of through TIM17:23 translocase in ftsh4-1 mutant suggests that a low Tim17-2 in isolated mitochondria. We observed a time-dependent molecular mass Tim17-2 complex, which is present in elevated decrease in Tim17-2 amounts in wild-type mitochondria, while the amounts in mutant mitochondria, could represent a functional levels of this protein remained stable in mitochondria isolated from translocase involved in import of preproteins. On the other hand, the plants expressing proteolytically inactive FTSH4 (Fig. 3B). TIM17:23 translocase is a very dynamic complex in which its Moreover, we found that Tim17-2 specifically co-precipitates subunits alter their conformations and undergo association and with FTSH4(H486Y) (Fig. 3C,D). We conclude from these dissociation to facilitate preprotein import (Schulz et al., 2015). studies that Tim17-2 represents a bona fide substrate of the Therefore, we cannot rule out the possibility that the presence of FTSH4 protease. excessive amounts of Tim17-2 induces changes in the architecture and dynamics of TIM17:23 translocase that have a stimulatory impact on Lack of FTSH4 protease is associated with enhanced import of at least a specific set of preproteins. Noteworthy, Tim17-2 capacity for the import of model substrates for the TIM17:23 contains a C-terminal extension that protrudes into the outer translocase mitochondrial membrane providing a physical link between the An imbalance in levels of different complex subunits might lead to inner and outer mitochondrial membranes (Murcha et al., 2005). Thus, changes in the activity of the whole protein complex (König et al., accumulation of Tim17-2 in the ftsh4 mutant could enhance the 2016). Since our results suggest that FTSH4 protease controls tethering between mitochondrial membranes facilitating preprotein Tim17-2 levels and by this modulates the architecture of TIM17:23 import through the TIM17:23 complex (Donzeau et al., 2000). translocase, we questioned how the loss of FTSH4 influences However, the molecular mechanism detailing how the Tim17-2 levels mitochondrial protein import. To establish the role of FTSH4 in this control the rate of preprotein import into the plant mitochondria process, we analyzed the capacity for in vitro uptake of the remains to be elucidated. alternative oxidase (AOX, Genbank X68702), a precursor known to be imported via TIM17:23 translocase (Murcha et al., 2005), into Conclusions ftsh4-1 mutant mitochondria (Fig. 4A). We found that mitochondria In conclusion, our report suggests that the FTSH4 protease has a devoid of FTSH4 protease display a substantially increased rate of novel role of in the regulation of preprotein influx into the import for the radiolabeled AOX precursor. In contrast, in vitro mitochondria by regulating the abundance of Tim17-2 levels. These uptake of the adenine nucleotide transporter (ANT) (Murcha et al., findings provide new insights into the significance of ATP- 2005), a precursor known to be imported via the TIM22 translocase dependent proteolysis in the maintenance of mitochondrial complex, showed no difference in the rate of import in mitochondria proteostasis in plants. isolated from ftsh4-1 compared to in wild type (Fig. 4B). Furthermore, we found that overexpression of FTSH4 protease in MATERIALS AND METHODS a ftsh4-1 background led not only to a reduction in Tim17-2 levels Reagents (Fig. S2), but also to a decrease in the rate of AOX import (Fig. S4). A detailed list of reagents used in this study is provided in Table S1. This is in line with previous findings, where the mammalian homolog of FTSH4, YME1L, was shown to downregulate the Plant material and growth conditions activity of TIM17:23 translocase by mediating the proteolysis of Arabidopsis thaliana lines used were: ecotype Col-0 wild type, T-DNA

Tim17A isoform (Rainbolt et al., 2013). Increased import capacity insertion lines ftsh4-1 (SALK_035107/TAIR) and ftsh4-2 (GABI_103H09/ Journal of Cell Science

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TAIR) (Gibala et al., 2009), the ftsh4-1 FTSH4 complementation line and Co-immunoprecipitation ftsh4-1 FTSH4(H486Y) mutant line, and were grown on 0.5× MS medium In order to perform immunoprecipitation of FLAG-tagged FTSH4(H486Y) supplemented with 3% (w/v) sucrose in chambers in a 16 h light and 8 h protein, mitochondria were resuspended in digitonin solubilization buffer dark (long-day photoperiod) at 22°C for 2 weeks with a light intensity of (1% digitonin, 20 mM Tris-HCl, 0.1 mM EDTA, 100 mM NaCl, 10% 150 μmol m−2 s−1. To test the morphological phenotype of A. thaliana lines, glycerol, pH 7.7) at 1 mg/ml. PMSF and EDTA-free protease inhibitor plants were grown in soil in a climate-controlled chamber in a 8 h light and cocktail were added and samples were incubated for 30 min at 4°C with 16 h dark (short-day photoperiod) at 22°C for 10 weeks. mixing. After a clarifying centrifugation (18,000 g for 15 min), solubilized material was loaded on anti-FLAG affinity matrix and incubated under Generation of ftsh4-1 FTSH4 complementation line constant rotation for 1.5 h at 4°C. After excessive washing steps proteins FTSH4 cDNA sequence was amplified using primers FTSH4.FLAGfor and were eluted and subjected to SDS-PAGE and immunoblot analysis. FTSH4.FLAGrev (Table S2) and cloned into pENTR™/D-TOPO Immunoprecipitation assays using antibodies raised against Tim17-2 and (Invitrogen). The resulting plasmid, FTSH4.FLAG pENTR, was used in a Protein-A–Sepharose resin were performed as described previously (Wang gateway LR recombination reaction with destination vector pGWB514, and et al., 2012). a final construct, FTSH4.FLAG pGWB514, was obtained. The ftsh4-1 FTSH4 complementation line was made by the Agrobacterium tumefaciens- Acknowledgements mediated transformation (Bernhardt et al., 2012). We thank Tsuyoshi Nakagawa (Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, Japan) for vector pGWB514 and Lee Sweetlove (Department of Plant Sciences, University of Oxford, ftsh4-1 Generation of the FTSH4(H486Y) mutant line UK) for Slp1 antibodies. In order to abolish the proteolytic activity of FTSH4 protease, a highly 2+ H conserved histidine at the position 486 (a Zn -binding site, in the ExxH Competing interests motif) was mutated into a tyrosine residue (Westphal et al., 2012). The The authors declare no competing or financial interests. mutation was introduced on FTSH4.FLAG pENTR plasmid using a QuikChange Lightening site-directed mutagenesis kit (Agilent Author contributions Technologies) and primers Mut_prot_H_FP and Mut_prot_H_RP M.O. and H.J. designed the study. M.O. and K.P. performed the experiments. (Table S2) resulting in a FTSH4(HIS).FLAG pENTR plasmid. Next, the M.W.M. provided the materials. All authors analyzed the data. M.O. wrote the Gateway LR recombinant reaction was performed with a FTSH4(HIS). manuscript with contribution from other authors. FLAG pENTR plasmid and destination vector pGWB514. This resulted in the construction of a final plasmid, FTSH4(H486Y).FLAG pGWB514, Funding bearing FTSH4(H486Y).FLAG under the constitutive CaMV 35S This work was supported by a grant from the National Science Centre (Narodowe Centrum Nauki, NCN) (2015/16/S/NZ3/00364) awarded to M.O. M.W.M. is promotor. ftsh4-1 FTSH4(H486Y) line was made by the Agrobacterium supported by an Australian Research Council Future Fellowship (FT13100112). tumefaciens-mediated transformation (Bernhardt et al., 2012). Supplementary information Immunoblot analysis Supplementary information available online at Selected proteins were probed with the specific antibodies indicated in http://jcs.biologists.org/lookup/doi/10.1242/jcs.200733.supplemental Table S3. 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