Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Lon protease inactivation in Drosophila causes unfolded protein stress and inhibition of mitochondrial translation

Lon protease inactivation in Drosophila causes unfolded protein stress and inhibition of... Mitochondrial dysfunction is a frequent participant in common diseases and a principal suspect in aging. To combat mitochondrial dysfunction, eukaryotes have evolved a large repertoire of quality control mechanisms. One such mechanism involves the selective degradation of damaged or misfolded mitochondrial proteins by mitochondrial resident proteases, including proteases of the ATPase Associated with diverse cellular Activities (AAA ) family. The importance of the AAA family of mitochondrial proteases is exemplified by the fact that mutations that impair their functions cause a variety of human diseases, yet our knowledge of the cellular responses to their inactivation is limited. To address this matter, we created and characterized flies with complete or partial inactivation of the Drosophila matrix-localized AAA protease Lon. We found that a Lon null allele confers early larval lethality and that severely reducing Lon expression using RNAi results in shortened lifespan, locomotor impairment, and respiratory defects specific to respiratory chain complexes that contain mitochondrially encoded subunits. The respiratory chain KD defects of Lon knockdown (Lon ) flies appeared to result from severely reduced translation of mitochondrially encoded genes. This translational defect was not a consequence of reduced mitochondrial transcription, as evidenced KD by the fact that mitochondrial transcripts were elevated in abundance in Lon flies. Rather, the translational defect of KD Lon flies appeared to be derived from sequestration of mitochondrially encoded transcripts in highly dense KD ribonucleoparticles. The translational defect of Lon flies was also accompanied by a substantial increase in unfolded mitochondrial proteins. Together, our findings suggest that the accumulation of unfolded mitochondrial proteins triggers a stress response that culminates in the inhibition of mitochondrial translation. Our work provides a foundation to explore the underlying molecular mechanisms. Introduction stoichiometric imbalance between mitochondrial and Mitochondria are responsible for most of the energy nuclear encoded respiratory chain subunits can cause produced by a cell, but the generation of reactive oxygen misfolding and aggregation of the unassembled pro- 4,5 species (ROS) as a byproduct of this activity can damage teins . Fortunately, there are many surveillance pathways 1–3 + mitochondrial proteins, lipids, and DNA . Also, while that oppose or reverse this damage, including the AAA 6–8 + mitochondria contain their own genome, most mito- family of mitochondrial proteases . All of the AAA chondrial proteins are encoded in the nucleus, and a proteases form multimeric protein complexes and use ATP to unfold and transport substrates to an internal proteolytic cavity for degradation. In higher eukaryotes, there are five major mitochondrial AAA proteases that Correspondence: Leo J. Pallanck (pallanck@uw.edu) Department of Genome Sciences, University of Washington, 3720 15th are distinguished by their subunit composition and Avenue NE, Seattle, WA 98195, USA mitochondrial localization. The most well-studied mem- Department of Biochemistry, University of Washington, 1705 NE Pacific St., ber of the AAA protease family is Lon. Seattle, WA 98195, USA Edited by I. Amelio © 2018 The Author(s) Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Pareek et al. Cell Death Discovery (2019) 5:51 Page 2 of 14 Previous work indicates that Lon possesses three dif- an unfolded protein stress response that inhibits mito- ferent activities, serving as a chaperone and DNA-binding chondrial translation. 9,10 protein in addition to its role in proteolysis . Lon expression is regulated by multiple cellular stresses, Results including ROS and unfolded protein stress, and there is Creation of Lon-deficient Drosophila strains substantial support for the role of Lon in the degradation To explore the biological roles of Lon protease we used 11,12 of oxidatively damaged and misfolded proteins . CRISPR/Cas9 technology to create a null allele of the Although few Lon substrates are known with certainty, a Drosophila Lon gene (CG8798). Briefly, we constructed number of candidate Lon substrates have been identified guide RNAs designed to create double-strand breaks from biochemical studies aimed at the identification of flanking the Lon coding sequence (Suppl. Figure 1). We Lon-binding proteins, including the mitochondrial DNA also created a donor vector construct consisting of the (mtDNA) replication factors Twinkle, polymerase gamma, DsRed marker flanked by 5′ and 3′ untranslated sequences 13–19 Tfam, and the chaperones Hsp60 and mtHsp70 . from the Lon coding region for use in homology-directed Subsequent studies aimed at validating the significance of recombinational repair of the double-strand breaks. Flies these binding interactions indicated that Lon inactivation expressing the DsRed marker were then subjected to results in increased mtDNA copy number and destabili- whole-genome sequencing to verify correct targeting of zation of Hsp60 and mtHsp70 under environmental DsRed to the Lon locus and complete deletion of the Lon 20–22 stress . However, it is unclear whether these findings gene. Flies heterozygous for this deletion were fully viable reflect direct effects of Lon inactivation, or downstream with no detectable phenotypes. However, homozygotes cellular responses to loss of Lon activity. Moreover, died at the second instar larval stage of development, mutations in Lon have been shown to result in the demonstrating that Lon is essential for viability (Suppl. recessive developmental disorder CODAS (cerebral, Figure 2a). ocular, dental, auricular, and skeletal) syndrome, yet the RNAi often reduces but does not completely eliminate mechanisms by which mutations in Lon cause this disease target gene expression. Thus, we tested whether RNAi are currently unknown . lines targeting Lon would circumvent the lethality con- To create a simple, genetically tractable model system ferred by a null mutation in Lon. We tested two different to explore the biological role of Lon and the pathological RNAi constructs targeting Lon (designated Lon-RNAi-1 consequences of Lon inactivation, we used CRISPR/Cas9- and Lon-RNAi-2) that we used in previously published mediated gene targeting and RNAi to create Drosophila work to explore the influence of Lon on the abundance strains with complete and partial loss of Lon function. We and activity of the mitophagy factor PINK1 . Our pre- found that Lon is an essential gene in Drosophila and that vious work demonstrated that these RNAi lines differed in flies expressing RNAi against Lon (Lon knockdown flies, the efficiency by which they reduced Lon expression, with KD or Lon flies) had shortened lifespan, defective locomo- the Lon-RNAi-2 line resulting in greater reduction in Lon tion, and altered respiratory chain activity. The respiratory expression. We expressed each RNAi line using two GAL4 KD chain deficits in Lon flies were specific to respiratory drivers simultaneously: the pan-neuronal elav-GAL4 dri- chain complexes that contain subunits encoded by the ver and the ubiquitous da-GAL4 driver. Driving the mitochondrial genome, suggesting that altered expression stronger Lon-RNAi-2 line with this combination of GAL4 of mitochondrially encoded components underlies this drivers failed to yield viable adult flies, but flies expressing defect. Consistent with this conclusion, we found that the weaker Lon-RNAi-1 transgene were fully viable and reduced mitochondrial complex activity is accompanied fertile as young adults, and had no obvious morphological by reduced complex abundance and diminished mito- alterations. Western blot analyses performed on heads KD chondrial translation. The translational defect of Lon and whole flies indicated that Lon expression was sig- flies was not a consequence of reduced mitochondrial nificantly reduced in flies bearing the UAS-Lon-RNAi-1, transcription, but rather appeared to be a consequence of elav-GAL4, and da-GAL4 transgenes compared to con- reduced association of mitochondrial transcripts on trols expressing RNAi against the exogenous mCherry mitochondrial ribosomes and packaging of mitochondrial sequence (UAS-mCherry-RNAi) (Fig. 1a and Suppl. Fig- transcripts into highly dense ribonucleoparticles. The ure 2b). Flies bearing the Lon-RNAi-1, elav-GAL4, and da- KD translational defect of Lon flies was also accompanied GAL4 transgenes were used in most of the remaining KD by elevated abundance of unfolded mitochondrial pro- studies and will hereafter be called Lon . teins, and overexpression of another matrix protease, ClpP, partially rescued the defects associated with Lon Partial inactivation of Lon results in shortened lifespan, inactivation, possibly by reducing the burden of unfolded locomotor defects, and altered respiratory chain activity KD mitochondrial proteins. Together, our findings strongly To further characterize the Lon phenotypes, we suggest that Lon inactivation results in the activation of subjected the flies to several standard Drosophila Official journal of the Cell Death Differentiation Association Pareek et al. Cell Death Discovery (2019) 5:51 Page 3 of 14 a b c d kDa 8 1.0 ** α Lon *** 0.8 α Actin 0.6 0.4 1.2 *** 1.0 2 0.2 0.8 0.6 Days 0.4 0.2 Fig. 1 Inactivation of Lon results in shortened lifespan and defective locomotion. a Immunoblot analysis from heads of 1-day-old control and KD Lon flies using Lon and actin antibodies. The Lon band intensity is normalized against actin as a loading control. Significance was determined using KD Student’s t-test (***p< 0.0005 by Student’s t-test). The experiment was repeated at least three times. b Kaplan–Meier survival curves of Lon flies KD (n = 167, 50% survival 31 days) and controls (n = 174, 50% survival 73 days) (****p < 0.0001 by Mantel–Cox log-rank test). c One-day-old Lon flies KD KD exhibit a climbing defect. Error bars represent SEM (n = 135 for Lon , 134 for control, ***p < 0.0005 by Student’s t-test). d One-day-old Lon flies exhibit a significant decrease in flight index (n = 6 independent groups of 10–15 animals, **p < 0.005 from Student’s t-test). Control= UAS-mCherry- KD RNAi driven by elav-GAL4 and da-GAL4. Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 KD behavioral assays. Although Lon flies appeared normal Figure 3). Together, our findings indicate that partial upon eclosion, they were significantly shorter-lived than inactivation of Lon results in a progressive age-dependent the control flies (Fig. 1b). The maximal and median life- decline in oxidative phosphorylation capacity, and that KD spans of Lon flies were similar to those of flies bearing this decline may underlie the shortened lifespan and + KD null mutations in genes encoding the other AAA mito- behavioral deficits of Lon flies. chondrial protease family members dYME1L and 25,26 KD SPG7 . Young Lon flies also exhibited defects in Lon knockdown results in increased mtDNA-encoded climbing and flight starting early in life (Fig. 1c, d). transcript abundance and reduced translation Inactivation of Lon in other model systems results in All of the respiratory chain complexes exhibiting 27 KD reduced respiratory chain activity . Thus, we tested reduced abundance and assembly in Lon flies (com- KD whether Lon flies exhibit similar respiratory chain plexes I, III, IV, and V) contain one or more subunits defects, using established biochemical assays to quantify encoded by mtDNA. Additionally, our BN-PAGE and in- KD respiratory chain activity in young (1 day) and old gel activity assays in Lon flies indicated that the ATP KD (3 weeks) Lon flies. We found that respiratory chain synthase F subunit containing subcomplex (consisting KD complexes I and IV had reduced activity in old Lon flies entirely of subunits encoded by nuclear DNA) accumu- compared to the controls, while young flies showed lated at the expense of the F subcomplex (which includes reduction only in complex I activity (Fig. 2a). However, two subunits encoded by mtDNA). By contrast, complex KD complex II activity was increased significantly in both II, which exhibited increased activity in Lon flies, con- KD young and old Lon flies. These alterations could result sists entirely of nuclear DNA–encoded components. from a functional change in respiratory chain activity or These findings led us to hypothesize that reduced mtDNA from altered respiratory chain abundance. To distinguish abundance, reduced transcription of mtDNA-encoded between these possibilities while simultaneously con- subunits, and/or reduced translation of mitochondrial firming and extending our biochemical studies, we transcripts might account for the decreased expression of assessed the abundance of assembled complexes and their complexes I, III, IV, and V. corresponding activities using blue native PAGE (BN- To begin to explore these hypotheses, we first compared 28 KD PAGE) and in-gel enzyme activity assays . This work mtDNA abundance in Lon flies and the controls. While revealed that complexes I, III, and IV all exhibited both increased mtDNA abundance in response to Lon inacti- KD 20 reduced activity and reduced abundance in Lon flies, vation has been reported , mtDNA copy number was KD whereas complex II exhibited an increase in activity unchanged in Lon flies (Fig. 3a). This is consistent with (Fig. 2b–d). Additionally, this work revealed the presence our previous finding of unchanged mtDNA abundance of a partially assembled but catalytically active F sub- when Lon was knocked down with elav-GAL4 alone . unit–containing subcomplex of ATP synthase (complex We next analyzed the steady-state levels of several mito- V) (Fig. 2b, e). These alterations were also accompanied chondrial transcripts by qRT-PCR. We found that levels KD by a reduction in ATP content in Lon flies (Suppl. of all transcripts analyzed, including 12S and 16S rRNA Official journal of the Cell Death Differentiation Association Fold Change Percent Survival Distance Climbed (cm) Flight Index Pareek et al. Cell Death Discovery (2019) 5:51 Page 4 of 14 Complex IV 2.0 1.0 Day 1 Day 21 Complex IV ns 0.8 1.5 0.6 Complex III Complex III ns 1.0 ns Complex I Complex II Complex II Complex I 0.4 ** ** ** 0.5 0.2 0 0 b c d e kDa kDa kDa kDa C(V) SCs 2 C(V) CI CV CI 720 C(V) CIII 720 720 * C(IV) /CIII CIV CII 242 CIV CII 146 146 66 66 kDa Citrate synthase Fig. 2 Lon knockdown flies have altered respiratory chain function and abundance. a The activity of respiratory chain complexes in control KD and Lon flies at 1 day (left panel) and 21 days (right panel) of age (n = 2 independent groups of 500 adult flies, *p < 0.05, **p < 0.005 from Student’s KD t-test). b BN-PAGE analysis of mitochondrial protein extracts from 21-day-old control and Lon flies. The red asterisk marks the location of KD the subcomplex containing F subunit of ATP synthase that is only detected in Lon flies. Immunoblot of citrate synthase (bottom panel) was used as a loading control. c In-gel activity of mitochondrial complexes I and IV isolated from 21-day-old adults. SCs here refer to the supercomplexes. d In- gel activity of mitochondrial complex II isolated from 21-day-old flies. e In-gel activity of mitochondrial complex V isolated from 21-day-old adults. KD The red asterisk denotes the location of the subcomplex containing F subunit of ATP synthase in Lon flies. Note that the protein molecular weight markers shown in (b) were used as reference to mark the gels in (c–e). For BN-PAGE and in-gel activity assays, the images shown are representative of KD two independent biological replicates. Control = UAS-mCherry-RNAi driven by elav-GAL4 and da-GAL4. Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 KD and several mRNAs, were increased in old Lon flies This finding may reflect the fact that mitochondrial pro- (Fig. 3b). Additionally, the transcript abundance of the teins are extremely long-lived and that there is thus a mitochondrial transcription-promoting factors TFAM decreased need for mitochondrial translation during the KD and mtTFB2 was unchanged in Lon flies (Suppl. Fig- adult stage of development. However, previous work has ure 4). Together, these findings indicate that the respira- detected robust labeling of mitochondrial proteins using KD tory chain defects in Lon flies do not derive from mitochondria obtained from larvae, so we repeated our in reduced mitochondrial gene dosage or reduced mito- organello labeling studies using mitochondria from third chondrial transcription. instar larvae . Our western blot analysis confirmed that KD KD To test whether Lon flies manifest a translational Lon expression was greatly reduced in Lon larvae defect, we used S-labeled methionine to perform in relative to the controls (Fig. 3c). This experiment revealed organello labeling of mitochondria prepared from adult a substantial decrease in de novo labeling of mitochon- KD flies. However, we detected very little labeling of mito- drial translation products in Lon larvae relative to the chondrial proteins in our control flies (Suppl. Figure 5). controls (Fig. 3d), suggesting that the decreased Official journal of the Cell Death Differentiation Association Normalized respiratory chain activity Pareek et al. Cell Death Discovery (2019) 5:51 Page 5 of 14 a b ns 1.0 ** ** *** ** 0.5 ** ** kDa kDa 96 Lon Actin ** 1.0 COX1 CYTB 0.5 COXIII COXII/ATP6 ND4L/ ATP8 Fig. 3 Lon knockdown flies exhibit increased mitochondrial transcript abundance and decreased mitochondrial translation. a Mitochondrial KD DNA abundance was compared in Lon and control flies using qPCR to compare the ratio of mtDNA-encoded mt:Cyt-b to that of nuclear-encoded Act79b (n = 3 independent groups of 40–45 fly heads). Statistical significance was determined using Student’s t-test. b qRT-PCR was used to quantify steady-state abundance of the indicated mitochondrial RNAs in 21-day-old adult fly heads. Mitochondrial RNA abundance was normalized to the abundance of the nuclear-encoded Act79b transcript (n = 3 independent groups of 40–45 fly heads). Error bars indicate mean ± SEM. Student’s t-test was applied, *p < 0.05, **p < 0.005, ***p < 0.0005. c Immunoblot analysis of third instar larvae to confirm knockdown of Lon using actin as a loading control. n = 3 independent groups of five third instar larvae. Significance was determined using Student’s t-test, **p < 0.005. d In organello translation was performed using mitochondria isolated from third instar larvae. Mitochondria were labeled by incubating with S-methionine for 1 h. Positions of individual mitochondrially encoded proteins are indicated (left panel). Coomassie-stained gel (right panel) was used as a loading control. KD Control= UAS-mCherry-RNAi driven by elav-GAL4 and da-GAL4. Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 KD abundance of complexes I, III, IV, and V in Lon flies is a Mitochondrial transcripts are packaged into untranslated KD direct consequence of a translational defect. The particles in Lon flies increased abundance of mtDNA-encoded transcripts and To investigate the mechanism by which inactivation of nuclear DNA–encoded complex II may represent com- Lon impairs mitochondrial translation, we assessed the pensatory responses to alleviate this translational defi- state of mitochondrial ribosome assembly by performing 31–33 ciency . sucrose density gradient sedimentation analyses. Official journal of the Cell Death Differentiation Association mtDNA/NucDNA ratio Fold Change KD Log Fold Change (Lon /control) 2 Pareek et al. Cell Death Discovery (2019) 5:51 Page 6 of 14 gradient were then subjected to qRT-PCR to quantify the distribution of the 12S and 16S mitochondrial ribosomal 12S control RNAs, which mark the small (28S) and large (39S) KD Lon 28S 39S 55S mitochondrial ribosomal subunits, respectively. Co- localization of the 12S and 16S mitochondrial ribosomal RNAs within the same fraction is diagnostic for fully assembled and actively translating mitochondrial ribo- 20 somes (55S). This analysis revealed that the abundance of 12S and 16S ribosomal RNAs in fully assembled mito- chondrial ribosome fractions (fraction 15–17) was KD decreased by 5.8% and 6.4%, respectively, in Lon flies relative to the controls (Fig. 4). The mild decrease in mitochondrial ribosome assembly appeared insufficient to account for the translational 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 KD defect of Lon flies, especially in light of the fact that Fraction number mitochondrial transcripts are elevated in abundance relative to the controls. Thus, we examined other possible explanations for the translational defect of Lon-deficient 16S flies. One possible explanation of our findings was that control mitochondrial transcripts are translated at lower effi- KD KD 28S 39S 55S Lon ciency in Lon flies. To test this model, we used sucrose density gradient centrifugation to quantify the relative distribution of four different mitochondrial transcripts, including mt:ND5, mt:Cyt-b, mt:CoII, and ATPase8/6,in gradient fractions (Fig. 5). All of these mRNAs displayed a predominant sedimentation peak co-migrating with the KD 55S ribosome in control samples. By contrast, in Lon flies a smaller proportion of these transcripts co-migrated with the 55S ribosome, and this decrease was accom- panied by a striking increase in the proportion of these transcripts that sedimented to the bottom of the gradient 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 (Fig. 5). This finding suggests that a substantial proportion Fraction number KD of the mitochondrial transcripts in Lon flies are pack- aged into large untranslated particles. Fig. 4 Mitochondrial ribosome assembly is only mildly affected in KD KD Lon flies. Mitochondrial lysates from 21-day-old control and Lon Lon knockdown flies accumulate unfolded mitochondrial flies were subjected to sucrose density gradient fractionation to assess the state of assembly of mitochondrial ribosomes. The relative proteins and trigger the mitochondrial unfolded protein proportions of the small (28S) subunit, large (39S) subunit, and fully response assembled (55S) mitochondrial ribosomes were assessed by Lon is believed to play a critical role in the degradation subjecting the density gradient fractions to qRT-PCR using primer sets 34–36 of unfolded mitochondrial proteins . The accumula- specificto 12S rRNA, which marks the small subunit, and to 16S rRNA, tion of unfolded proteins in multiple cellular compart- which marks the large subunit. Co-localization of the 12S and 16S rRNAs is diagnostic of fully assembled and actively translating ments has been shown to activate a stress response known ribosomes. Fractions containing the small (28S) subunit, large as the unfolded protein response (UPR) that is specific (39S) subunit, and fully assembled (55S) ribosome are shaded cyan, to the compartment in which the unfolded proteins pink, and yellow, respectively. The relative abundance of a given rRNA 37–39 reside . The cytosolic and endoplasmic reticulum (ER) in each fraction was calculated as the percentage relative to the total UPR restore protein homeostasis through a two-tiered RNA abundance in all fractions after normalizing to a luciferase control RNA that was spiked into each of the fractions prior to RNA isolation. system consisting of increased expression of chaperones Control= UAS-mCherry-RNAi driven by elav-GAL4 and da-GAL4. to facilitate the refolding of misfolded proteins, and KD Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 phosphorylation-mediated inactivation of the cytosolic translation-initiation factor eIF2α to attenuate transla- tion . While previous work has established that unfolded KD Mitochondrial extracts from Lon flies and controls protein stress in the mitochondria triggers the induction were subjected to sucrose density gradient analysis as of chaperones and proteases, whether mitochondrial previously described . Fractions from the sucrose translation is inhibited by unfolded protein stress is less Official journal of the Cell Death Differentiation Association Relative abundance Relative abundance Pareek et al. Cell Death Discovery (2019) 5:51 Page 7 of 14 mt:ND5 ATPase8/6 control control KD KD Lon 28S 39S 55S 28S 39S 55S Lon 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Fraction number Fraction number control control mt:CoII mt:Cyt-b KD KD Lon Lon 28S 39S 55S 28S 39S 55S 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Fraction number Fraction number Fig. 5 Mitochondrial RNAs accumulate in large untranslated particles upon Lon knockdown. qRT-PCR analysis of individual sucrose gradient fractions was used to characterize the distribution of the indicated mtDNA-encoded mRNAs relative to the 28S and 39S subunits, and fully KD assembled 55S mitochondrial ribosomes. Mitochondrial homogenates for sucrose density fractionation were prepared from 21-day-old adult Lon and control flies. Relative abundance represents the fraction of the mRNA in any given fraction relative to the total after normalizing to a luciferase KD control RNA that was added to each of the fractions prior to RNA isolation. Control= UAS-mCherry-RNAi driven by elav-GAL4 and da-GAL4. Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 clear. Our findings that Lon inactivation appears to result to selected mitochondrial proteins to quantify the pro- in the sequestration of mitochondrially encoded mRNAs portion of each protein in the soluble and insoluble KD into translationally inactive particles, coupled with the fractions. Lon flies had higher levels of insoluble severe defect in mitochondrial translation, led us to mitochondrial proteins than the control flies at both day 1 hypothesize that the mitochondrial UPR triggers transla- and day 21 of age (Fig. 6a and Suppl. Figure 6a), indicating KD tional inhibition. that Lon flies accumulate unfolded mitochondrial pro- To explore our hypothesis we first analyzed whether teins. We next examined the abundance of the mito- KD KD Lon flies accumulate unfolded mitochondrial proteins chondrial UPR markers Hsp60 and Hsc70-5 in Lon and whether they activate the mitochondrial UPR. To test flies. This analysis revealed a marked increase in both KD whether unfolded mitochondrial proteins accumulate in Hsp60 and Hsc70-5 in Lon flies at day 1 and at day 21 KD Lon flies, we prepared fly head protein extracts using of age (Fig. 6b and Suppl. Figure 6b). Together, these KD Triton X-100. We then subjected Triton-soluble and findings indicate that Lon flies accumulate unfolded -insoluble proteins to western blot analysis using antisera proteins and trigger the mitochondrial UPR. Official journal of the Cell Death Differentiation Association Relative abundance Relative abundance Relative abundance Relative abundance Pareek et al. Cell Death Discovery (2019) 5:51 Page 8 of 14 4.5 kDa *** 3.5 ** 2.5 1.5 0.5 KD Lon aconitase compV PDH NDUFS3 3.5 ** 2.5 kDa 1.5 0.5 KD Lon Hsp60 Hsc70-5 Fig. 6 Inactivation of Lon protease results in the accumulation of unfolded mitochondrial proteins. a Triton-insoluble mitochondrial proteins KD detected by western blot in heads from 1-day-old control and Lon flies, using antibodies to complex Vβ, PDHα, aconitase, and NDUFS3. The results were quantified by ratio to actin and normalized to control levels. The experiment was repeated at least three times. *p< 0.05, **p< 0.005, ***p< 0.0005 by Student’s t-test. b Immunoblot analysis of head proteins using antibodies to Hsp60A and Hsc70-5. Protein was extracted from heads of day KD 1 control and Lon flies using RIPA buffer. Quantification was performed as in (a). Experiments were repeated at least three times. *p< 0.05, **p< KD 0.005 by Student’s t-test. Control = UAS-mCherry-RNAi driven by elav-GAL4 and da-GAL4. Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 Overexpression of ClpP protease ameliorates the defect of compared Lon knockdown flies overexpressing ClpP to Lon knockdown flies Lon knockdown flies expressing mCherry RNAi. How- KD Protein quality control in the mitochondrial matrix is ever, strong overexpression of ClpP in Lon flies wor- regulated by an elaborate network of proteases and cha- sened the locomotor defect (Suppl. Figure 7). Thus, we perones, including the AAA protease family member tested whether ClpP expression could rescue a climbing Clp protease. To test whether the accumulation of defect caused by expressing the Lon-RNAi-1 transgene KD KD-elav unfolded proteins in Lon flies is responsible for the using just the elav-GAL4 driver (designated as Lon ). phenotypes associated with Lon inactivation, we co- We first confirmed that driving the UAS-ClpP transgene expressed the proteolytic subunit of the Drosophila Clp with elav-GAL4 resulted in the production of detectable protease (CG5045, hereafter ClpP) along with the Lon- ClpP protein (Fig. 7a) and use of the elav-GAL4 driver in RNAi-1 RNAi construct and examined whether ClpP conjunction with the Lon-RNAi-1 transgene reduced Lon overexpression is capable of rescuing the locomotor expression (Suppl. Figure 8) and caused a climbing defect KD defect of Lon flies. To account for possible titration of (Fig. 7b). Results of this analysis revealed that ClpP GAL4 protein in the presence of two UAS transgenes, we overexpression rescued the locomotor defect of Lon Official journal of the Cell Death Differentiation Association Fold Change Insoluble Fraction Fold Change Pareek et al. Cell Death Discovery (2019) 5:51 Page 9 of 14 orders of magnitude greater than that of the nuclear genome . Moreover, expression of the mitochondrial and nuclear genomes must be coordinated to ensure proper stoichiometry of mitochondrial and nuclear-encoded kDa respiratory chain components. An imbalance in this coordination can result in the accumulation of misfolded 39 + proteins and mitochondrial dysfunction . The AAA Flag mitochondrial protease family is believed to play an important role in maintaining mitochondrial competence by degrading and thereby facilitating the replacement of Actin 43 oxidatively damaged and misfolded proteins. However, we know little of the cellular responses and the pathogenic mechanisms of diseases caused by mutations in the genes that encode the AAA proteases. Our current work advances our understanding of these matters by showing *** that inactivation of the AAA protease Lon results in **** respiratory chain defects that appear to result from translational inhibition. Our findings further indicate that the translational defect caused by Lon inactivation results at least in part from the sequestration of mtDNA-encoded mRNAs into translationally inactive particles. Finally, we find that insoluble mitochondrial matrix proteins accu- KD mulate in Lon animals, suggesting that mitochondrial protein aggregation triggers translational inactivation as a stress response. Our work provides a foundation to fur- ther explore the mechanisms by which mitochondrial unfolded protein stress inhibits mitochondrial translation. Protein unfolding in the ER and cytoplasm induce well- 37,38,40–43 characterized stress responses . Each of these stress pathways has two arms: increased expression of chaperones to refold proteins, and translational inhibition to limit further protein synthesis. Translational inhibition is mediated by phosphorylation of eIF2α and culminates in the formation of translationally stalled ribonucleopro- Fig. 7 ClpP overexpression rescues the climbing defect of Lon- tein/mRNA complexes known as stress granules . More deficient flies. a Immunoblot analysis of fly heads to confirm recently, work in a variety of experimental systems has expression of FLAG-tagged ClpP in flies bearing a UAS-ClpP-FLAG-HA established the existence of a mitochondria-specific ver- transgene and the elav-GAL4 pan-neuronal driver. b Climbing was mt 45–49 KD-elav sion of the unfolded protein response (UPR ) . measured in 1-day-old control flies (n = 94), Lon flies (n = 91), KD-elav Although the degree to which this pathway is evolutio- and Lon flies co-expressing ClpP protease (n = 83). Control = KD-elav UAS-mCherry-RNAi driven by elav-GAL4. Lon ; mCherry RNAi = UAS- narily conserved remains an active area of investigation, it KD-elav Lon-RNAi-1 and UAS-mCherry-RNAi driven by elav-GAL4. Lon ; UAS- has been shown in both vertebrate and invertebrate ClpP= UAS-Lon-RNAi-1 and UAS-ClpP-FLAG-HA driven by elav-GAL4. models that mitochondrial unfolded protein stress trig- Error bars represent SEM. ***p< 0.0005, ****p< 0.0001 by Student’s gers the transcriptional activation of genes involved in t-test 45,47 protein folding and degradation . However, previous mt work has not clearly established whether the UPR also knockdown flies, thus supporting the hypothesis that the results in inhibition of mitochondrial translation. Recently accumulation of unfolded proteins is responsible for the published findings in vertebrate cell culture have revealed phenotypes of Lon-deficient flies (Fig. 7b). that inactivation of the matrix chaperone Trap1 results in inhibition of mitochondrial translation as a consequence Discussion of a pre-RNA processing defect . Our findings that Lon Mitochondria are particularly prone to damage. They knockdown flies have both an excess of mitochondrial are the primary source and recipient of damaging ROS unfolded proteins and an impairment of mitochondrial mt and continual replication of the mitochondrial genome translation raise the possibility that the UPR , like other throughout life culminates in mutation frequencies often unfolded protein stress pathways, has a second arm Official journal of the Cell Death Differentiation Association Distance Climbed (cm) Pareek et al. Cell Death Discovery (2019) 5:51 Page 10 of 14 involving translational inhibition. However, the precise Materials and methods mechanism by which mitochondrial unfolded protein Fly stocks and maintenance stress in Lon-deficient animals inhibits mitochondrial Drosophila stocks were maintained on cornmeal- translation will require further investigation. molasses food at 25 °C on a 12 h:12 h light–dark cycle. Although most of the respiratory chain complexes The UAS-Lon-RNAi-1 construct P{GD14030}-v36036 was exhibited reduced activity and abundance upon Lon obtained from the Vienna Drosophila Resource Center. inactivation, complex II activity was increased. This The w , UAS-mCherry RNAi (P{VALIUM20-mCherry} increase may simply represent a compensatory response, attP2), UAS-Lon-RNAi-2 (P{TRiP.HMS01060}attP2), as complex II has no mitochondrially encoded subunits, elav-GAL4, and da-GAL4 driver lines were obtained from and increased complex II activity has previously been the Bloomington Stock Center (Bloomington, IN, USA). observed in mutants with defects in mitochondrial gene The UAS-ClpP-FLAG-HA transgenic line was obtained 30,51,52 expression . However, it is also possible that from the Fly Facility, National Centre for Biological Sci- increased complex II activity is a consequence of reduced ences, Bangalore, India. degradation of the complex II assembly factor Sdh5, given The Lon knockout allele was created using CRISPR/ that previous work has shown that Lon mediates the Cas9-mediated gene editing according to a published 53 25,57 turnover of Sdh5 . Lon knockdown might thus reduce procedure . Briefly, we replaced the Lon (CG8798) the degradation of Sdh5 and promote complex II assem- coding sequence with DsRed through homology-mediated bly. The increased abundance of mitochondrially encoded repair. The following primer sequences were used for transcripts upon Lon inactivation may likewise reflect guide RNAs targeting the 5′ and 3′ UTR regions of Lon: either a general compensatory response or a specific 5′-Guide RNA change due to altered turnover of a possible Lon sub- Sense oligo: 5′-CTTCGATAATCACTCACCACACAT strate. Increased mitochondrial transcript abundance has T-3′ been reported previously in mutants with defective Antisense oligo: 5′-AAACAATGTGTGGTGAGTGAT mitochondrial RNA processing and translation, suggest- TATC-3′ ing that increasing the abundance of mitochondrial 3′-Guide RNA mRNA is a common response to inadequate mitochon- Sense oligo: 5′-CTTCGGGGTGTTGCGGGTGTTGA 30,31,52 drial translation . However, the accumulation of T-3′ KD mitochondrially encoded RNAs in Lon flies could also Antisense oligo: 5′-AAACATCAACACCCGCAACAC CCC-3′ be explained by the finding that these flies accumulate the RNA-binding protein Lrpprc1 (leucine-rich penta- Sequences flanking the Lon coding region were ampli- tricopeptide repeat motif-containing 1 protein; Suppl. fied from genomic DNA to facilitate homology-directed Figure 9), which is known to stabilize mitochondrial repair using the following primer sequences: 51,54,55 transcripts . Finally, Lon is a known component of 5′-Homology arm mitochondrial nucleoids, and could therefore influence Forward: 5′-CCGGCACCTGCGGCCTCGCAGTGCT the turnover and abundance of other proteins involved in CCGATCACGTTGGGAATGGG-3′ the synthesis or stabilization of mitochondrial tran- Reverse: 5′-CCGGCACCTGCGGCCCTACGTGTGGT scripts . Future work will be required to address these GAGTGATTATGTGACGGCTGGTG-3′ matters. 3′-Homology arm There are many unanswered questions regarding the Forward: 5′-GGCCGCTCTTCATATGATAGGTTTTA biological roles of the AAA protease family and the TAAATATCTATCGTTATCAGG-3′ mitochondrial response to unfolded protein stress. For Reverse: 5′-CCGGGCTCTTCTGACTTTCCCCGCCT example, mutations in the human gene encoding Lon CACCGGTGGACGGCC-3′ cause a developmental disorder known as CODAS syn- Thesehomologyarmswerethenclonedintothe pHD- drome . However, the underlying mechanisms causing DsRed-attP vector containing the eye-specific 3xP3 disease are completely unknown. Although Lon is well promoter fused with DsRed and the resulting construct known to promote the degradation of oxidatively was microinjected into Cas9-expressing embryos by a damaged and unfolded proteins, the specific protein commercial service (Rainbow Transgenic Flies Inc.). substrates of Lon are largely unknown. Whether unfolded Flies bearing the Lon deletion were identified by protein stress in the mitochondria generally triggers screening the offspring of injected adults for expression translation inhibition, or this feature is specific to Lon of red fluorescence in the compound eye. Flies expres- inactivation is also unclear. Our study provides a foun- sing the DsRed marker were then further subjected to dation to address these questions and the mechanisms whole-genome sequencing to verify deletion of the Lon underlying CODAS syndrome in future work. gene. Official journal of the Cell Death Differentiation Association Pareek et al. Cell Death Discovery (2019) 5:51 Page 11 of 14 Lifespan and behavioral analyses the oxidation of NADH at 340 nm using ubiquinone-1 Lifespan and behavioral analyses were performed using as an electron acceptor. Nonspecific activity was deter- male flies. Age in all these experiments refers to the mined using 10 μM rotenone and subtracted to calculate number of days following eclosion. Longevity assays were complex I-specific activity. Complex II activity was performed at 25 °C and involved 20 flies per vial. Flies determined by monitoring the reduction of 2,6-dichlor- were transferred to fresh food every 2–3 days and the ophenolindophenol at 600 nm in the presence of succi- nate and decylubiquinone. Background activity was number of dead flies was recorded during each transfer. Kaplan–Meier lifespan curves were generated using determined using 10 mM malonate and subtracted to GraphPad Prism v5, and we used the Mantel–Cox log- calculate complex II-specific activity. Complex III activity rank test to determine the statistical significance of dif- was determined by monitoring the reduction of cyto- ferences in survival between tested genotypes. chrome c at 550 nm in a reaction mixture containing For climbing and flight assays, flies were anesthetized decylubiquinol and cytochrome c. Nonspecific activity with CO and allowed to recover for at least 24 h before was determined using antimycin A and subtracted to the experiment. Climbing behavior was assessed using the calculate complex III-specific activity. Complex IV activity Rapid Iterative Negative Geotaxis (RING) assay at day 1 assay was performed by monitoring the oxidation of according to a previously published protocol with minor reduced cytochrome c at 550 nm. Background activity was modifications . In particular, height climbed was mea- determined using potassium cyanide and used to calculate sured on still images from video recordings rather than on complex IV-specific activity. All activities were normal- photographs. Briefly, 15 flies were transferred into plastic ized to citrate synthase activity, which was determined by vials and these vials were then loaded onto the RING following the reduction of 5,5′-dithiobis (2-nitrobenzoic apparatus. The apparatus was tapped down to initiate the acid) at 412 nm in the presence of acetyl-coenzyme A and climbing response and the height climbed by each fly after oxaloacetate. 3 s was recorded. The climbing assay was repeated three times for each group. Total ATP determination Flight assays were performed according to a previously Total ATP was determined from whole flies according 59,60 61 published protocol . Briefly, an acetate sheet was to a previously published procedure . Briefly, five 21-day- divided into five equal parts, coated with grease and old flies were homogenized in 100 μL of homogenization inserted into a 2-liter graduated cylinder. One-day-old buffer (6 M guanidine HCL, 100 mM Tris (pH 7.8), and 4 flies were tapped into a funnel at the top of the cylinder mM EDTA). The samples were boiled for 5 min and and became stuck to the vacuum grease where they subjected to centrifugation for 3 min at 21,000 g to alighted. The number of flies that alighted in each of the remove debris. The supernatant was then diluted to 1:750 five sections was counted and multiplied by the number and total ATP content was measured using an ATP corresponding to each section (0-4, labeled from bottom determination kit (A22066, Molecular Probes). Total ATP to top). The flight index was calculated by summing these content was determined by comparing the luminescence values and dividing this sum by the maximum possible measurements for each sample to the ATP standard curve score (four times the number of flies used in the assay). At and normalized to the total number of flies used in the least 100 flies of each genotype were used for a given assay. experiment. Blue native PAGE (BN-PAGE) analysis and in-gel activity Mitochondrial respiratory chain activity assay assay Mitochondrial respiratory chain activity assays were BN-PAGE and in-gel activity assays were performed performed according to a published procedure with sev- according to a previously published protocol . Briefly, eral minor modifications . Briefly, 1000 adult flies were 100 μg of mitochondria prepared from 3-week-old adult homogenized in isolation buffer (5 mM Tris (pH 7.4), flies was solubilized in a buffer containing a digitonin/ 250 mM sucrose, and 2 mM EGTA) with 1% (w/v) fatty protein (w/w) ratio of 8 and subjected to centrifugation at acid free bovine serum albumin. The lysate was subjected 20,000 g for 10 min at 4 °C. Coomassie G-250 was added to centrifugation at 600 g for 10 min to remove cellular to the supernatant and the sample was analyzed by native debris. The supernatant was then subjected to further PAGE. Following electrophoresis, the resulting gel was centrifugation at 7000 g for 10 min to pellet mitochondria. subjected to in-gel activity assays as described below. Mitochondria were washed twice in isolation buffer, The combined complex I and IV in-gel activity assay resuspended in the same buffer, flash frozen in liquid was performed by first incubating the gel in a solution nitrogen, and stored at −80 °C. Roughly 100 µg of mito- containing 1 mg/ml of the complex IV substrate cyto- chondria was used for each assay. Complex I activity was chrome c along with 0.5 mg/ml 3,3′-diaminobenzidine determined spectrophotometrically by monitoring and 45 mM phosphate buffer (pH 7.4) for 40 min. After Official journal of the Cell Death Differentiation Association Pareek et al. Cell Death Discovery (2019) 5:51 Page 12 of 14 the appearance of brown reaction products, the gel was the DNeasy Blood & Tissue kit (Qiagen). A total 5 ng of washed with water and incubated in a solution containing genomic DNA was used as a template to perform qPCR 0.1 mg/ml of the complex I substrate NADH along with using iTaq Universal SYBR Green Supermix (Bio-Rad). 2 mM Tris (pH 7.4) and 2.5 mg/ml nitrotetrazolium blue Mitochondrial DNA levels were estimated by using pri- chloride for 20 min. The reaction was quenched with 10% mers (Supplemental Table 1) to amplify the mt:Cyt-b acetic acid upon the appearance of the violet color indi- gene, and normalized to levels of the nuclear gene Act79b. The relative fold change was determined through the cative of complex I activity. −ΔΔCt 30 The complex II in-gel activity assay was performed by 2 method . incubating the gel in a solution consisting of 5 mM Tris (pH 7.4), 20 mM sodium succinate, 2.5 mg/ml nitrote- RNA isolation and quantitative reverse transcription-PCR trazolium blue chloride, and 0.2 mM phenazine metho- (qRT-PCR) sulfate for 40 min. The reaction was quenched with 10% RNA isolation and qRT-PCR was performed according 24,30,51 acetic acid upon the appearance of the violet color indi- to a previously published procedure . Total RNA cative of complex II activity. was isolated from 40–50 heads obtained from 21-day-old The complex V in-gel activity assay was carried out by adult flies using the Direct-zol RNA MiniPrep kit (Zymo incubating the gel in a solution containing 35 mM Tris, Research). The RNA was reverse transcribed to cDNA 270 mM glycine, 14 mM magnesium sulfate, 10 mM ade- using the iScript cDNA Synthesis Kit (Bio-Rad). For RNA nosine triphosphate, and 0.2% lead (II) nitrate for 16 h. quantification, qRT-PCR experiments were performed The reaction was stopped using 50% methanol upon the using iTaq Universal SYBR Green Supermix and a appearance of silver bands indicative of complex V activity. LightCycler 480 (Roche). Each sample was analyzed in triplicate and normalized to Act79b transcript abundance. −ΔΔCt Immunoblotting The relative fold change was determined by the 2 Fly heads were homogenized inRIPA bufferwithprotease method. All primers used for qRT-PCR are listed in inhibitor cocktail (Roche) for 20 min on ice, and after Supplemental Table 1. centrifugation at 21,000 × g for 20 min the supernatant was collected and subjected to western blot analysis. The anti- In organello translation bodies used were as follows: rabbit anti-LONP1 1:500 De novo labeling of mitochondrial translation products 30,51 (NBP1-81734, Novus Biologicals); mouse anti-Actin was performed as previously described . Approxi- 1:50,000 (MAB1501, Chemicon/Bioscience Research mately 750 µg of mitochondria isolated from third instar Reagents); mouse anti-FLAG 1:1000 (F3165, Sigma); rabbit larvae or adult flies was resuspended in 500 µl of trans- anti-citrate synthase 1:1000 (CISY11-A, Alpha Diagnostics), lation buffer (100 mM mannitol, 10 mM sodium succi- rabbit anti-Hsp60 1:500 (D307, Cell Signaling Technology); nate, 80 mM potassium chloride, 5 mM magnesium and rabbit anti-GRP 75 (H155) 1:1000 (sc-13967, Santa chloride, 1 mM potassium phosphate, 25 mM HEPES (pH Cruz) in Phosphate buffered saline with 0.1% Tween-20 . 7.4), 60 μg/ml all amino acids except methionine, 5 mM The secondary antibodies (anti-mouse HRP and anti-rabbit ATP, 0.2 mM GTP, 6 mM creatine phosphate, and 60 μg/ HRP (Sigma)) were used at a dilution of 1:10,000. Western mL creatine kinase) supplemented with 500 μCi/ml blot images were quantified using ImageJ software (NIH) of S-methionine (Perkin–Elmer). Following incubation and normalized to actin. Each experiment was performed in at 30 °C for 1 h, mitochondria were washed four times triplicate. using isolation buffer and resuspended in SDS sample To analyze unfolded mitochondrial proteins, protein buffer. Roughly 300 µg of mitochondria was subjected to was extracted from fly heads as previously described, SDS-PAGE. Following electrophoresis, the gel was dried except that 0.5% rather than 1% Triton X-100 was used in and exposed to a phosphor screen. The phosphor screen accordance with other work on mitochondrial pro- was scanned using a gel imaging scanner (GE Typhoon 63,64 teins . Antibodies for assaying mitochondrial protein FLA 9000). Mitochondrial translation profile was com- folding status were used as follows: mouse anti-NDUFS3 pared to a previously published study done in Drosophila 1:500 (ab14711, Abcam), mouse anti-Complex V Beta larvae . Roughly 75 µg of mitochondria was loaded on gel 1:2000 (A-21351, Invitrogen), mouse anti-PDH E1α and stained with Coomassie to use as a loading control. (clone 8D10E6) 1:1000 (45-660-0, Fisher Scientific), and rabbit anti-aconitase (ACO2) 1:2000 (AP1936C, Abgent). Mitochondrial ribosomal profiling using sucrose density gradient assay Genomic DNA isolation and mitochondrial DNA copy Mitochondrial ribosomal profiling was performed number estimation according to a previously published procedure with minor Mitochondrial and nuclear DNA was isolated from modifications . Freshly isolated mitochondria (2 mg) were 40–50 heads obtained from 21-day-old adult flies using incubated on ice in lysis buffer (260 mM sucrose, 100 mM Official journal of the Cell Death Differentiation Association Pareek et al. Cell Death Discovery (2019) 5:51 Page 13 of 14 NH Cl, 10 mM MgCl , 30 mM Tris-HCl pH 7.5, 40 U/ml 7. Rugarli, E. I. & Langer, T. Mitochondrial quality control: a matter of life and 4 2 death for neurons. EMBO J. 31, 1336–1349 (2012). Protector RNase Inhibitor, and 1% Triton X-100) supple- 8. Quiros,P.M., Langer, T. & Lopez-Otin,C.New roles for mitochondrialproteases mented with EDTA-free complete protease inhibitor in health, ageing and disease. Nat. Rev. Mol. Cell Biol. 16,345–359 (2015). cocktail (Roche) and PhosSTOP phosphatase inhibitor 9. Pinti, M. et al. Emerging role of Lon protease as a master regulator of mito- chondrial functions. Biochim. Biophys. Acta 1857, 1300–1306 (2016). cocktail (Roche). Mitochondrial lysates were cleared by 10. Pinti, M. et al. Mitochondrial Lon protease at the crossroads of oxidative stress, pelleting the debris through centrifugation at 10,000 g for ageing and cancer. Cell.Mol.LifeSci. 72,4807–4824 (2015). 11. Nystrom, T. Role of oxidative carbonylation in protein quality control and 45 min at 4 °C. The supernatant was then loaded onto a senescence. EMBO J. 24,1311–1317 (2005). 7–47% linear sucrose gradient made in a buffer containing 12. Bota, D. A. & Davies, K. J. Lon protease preferentially degrades oxidized 100 mM NH Cl, 10 mM MgCl , 30 mM Tris-HCl pH 7.5, 4 2 mitochondrial aconitase by an ATP-stimulated mechanism. Nat. Cell Biol. 4, and EDTA-free complete protease inhibitor cocktail 674–680 (2002). 13. Wagner, I.,Arlt, H.,van Dyck,L., Langer,T.& Neupert, W. Molecularchaperones (Roche). Samples were then subjected to centrifugation at cooperate with PIM1 protease in the degradation of misfolded proteins in 39,000 rpm for 6 h at 4 °C. Fractions of 500 μl from the mitochondria. EMBO J. 13,5135–5145 (1994). sucrose gradient sedimentation were collected and RNA 14. Savel’ev, A. S. et al. ATP-dependent proteolysis in mitochondria. m-AAA pro- tease and PIM1 protease exert overlapping substrate specificities and coop- was extracted from each fraction using TRIzol LS Reagent eratewiththe mtHsp70system. J. Biol. Chem. 273,20596–20602 (1998). (Invitrogen) and the Direct-zol RNA MiniPrep kit. RNA 15. Major, T., von Janowsky, B., Ruppert, T., Mogk, A. & Voos, W. Proteomic analysis was reverse transcribed and qRT-PCR analyses were of mitochondrial protein turnover: identification of novel substrate proteins of the matrix protease pim1. Mol. Cell Biol. 26,762–776 (2006). performed to quantify RNAs as described above. 16. Bayot, A. et al. Identification of novel oxidized protein substrates and phy- siological partners of the mitochondrial ATP-dependent Lon-like protease Statistics Pim1. J. Biol. Chem. 285,11445–11457 (2010). 17. Li, L. et al. Changes in specific protein degradation rates in Arabidopsis All findings are presented as mean ± SEM. Unless thaliana reveal multiple roles of Lon1 in mitochondrial protein homeostasis. otherwise stated, statistical significance was calculated Plant J.: Cell Mol. Biol. 89,458–471 (2017). using two-tailed Student’s t-test in GraphPad Prism v5. 18. Song, J. Y.,Marszalek,J.&Craig, E. A. Cysteine desulfurase Nfs1and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold. Proc. Natl Acad. Sci. USA 109,10370–10375 (2012). Acknowledgements 19. Ciesielski, S. J.,Schilke,B., Marszalek, J. &Craig,E.A.Protectionofscaffold We thank Dr. Scott Kennedy (Department of Pathology, University of del protein Isu from degradation by the Lon protease Pim1 as a component of Washington) for assistance with whole genome sequencing of the Lon Fe-S cluster biogenesis regulation. Mol. Biol. Cell. 27,1060–1068 (2016). mutant; Dr. Dana Miller for allowing us to use her lab space for experiments 20. Matsushima, Y., Goto, Y. & Kaguni, L. S. Mitochondrial Lon protease regulates involving radioactive isotopes; and Dr. Evan Eichler, Dr. Christine Queitsch, and mitochondrial DNA copy number and transcription by selective degradation Dr. Stanley Fields for providing access to their lab facilities. We thank Dr. Paul of mitochondrial transcription factor A (TFAM). Proc. Natl Acad. Sci. USA 107, M. Macdonald for providing an antibody against Lrpprc protein. This work was 18410–18415 (2010). supported by a grant from the National Institutes of Health to LP 21. Lu, B. et al. Phosphorylation of human TFAM in mitochondria impairs DNA (R21NS094901). binding and promotes degradation by the AAA+Lon protease. Mol. Cell 49, 121–132 (2013). Conflict of interest 22. Kao, T. Y. et al. Mitochondrial Lon regulates apoptosis through the association The authors declare that they have no conflict of interest. with Hsp60-mtHsp70 complex. Cell Death Dis. 6, e1642 (2015). 23. Strauss, K. A. et al. CODAS syndrome is associated with mutations of LONP1, encoding mitochondrial AAA+Lon protease. Am.J.Hum. Genet. 96, 121–135 Publisher's note (2015). Springer Nature remains neutral with regard to jurisdictional claims in 24. Thomas, R. E.,Andrews,L.A., Burman, J.L., Lin, W. Y. &Pallanck, L. J. PINK1- published maps and institutional affiliations. Parkin pathway activity is regulated by degradation of PINK1 in the mito- chondrial matrix. PLoS Genet. 10, e1004279 (2014). The online version of this article (https://doi.org/10.1038/s41420-018-0110-1) 25. Pareek, G., Thomas, R. E. & Pallanck, L. J. Loss of the Drosophila m-AAA contains supplementary material, which is available to authorized users. mitochondrial protease paraplegin results in mitochondrial dysfunction, shortened lifespan, and neuronal and muscular degeneration. Cell death & Dis. 9, 304 (2018). Received: 7 September 2018 Accepted: 11 September 2018 26. Qi, Y.,Liu,H., Daniels, M. P.,Zhang,G.&Xu,H. Loss ofDrosophila i-AAA protease, dYME1L, causes abnormal mitochondria and apoptotic degenera- tion. Cell Death Differ. 23,291–302 (2016). 27. Quiros, P. M. et al. ATP-dependent Lon protease controls tumor bioenergetics References by reprogramming mitochondrial activity. Cell Rep. 8,542–556 (2014). 1. Kauppila,T.E.S., Kauppila,J.H.K.&Larsson, N. G. Mammalianmitochondria 28. Garcia, C. J.,Khajeh, J.,Coulanges,E., Chen,E. I.&Owusu-Ansah, E. Regulation and aging: an update. Cell Metab. 25,57–71 (2017). of mitochondrial complex I biogenesis in Drosophila flight muscles. Cell Rep. 2. Sun, N., Youle,R.J.& Finkel, T.The mitochondrialbasis of aging. Mol. Cell 61, 20,264–278 (2017). 654–666 (2016). 29. Vincow, E. S. et al. The PINK1-Parkin pathway promotes both mitophagy and 3. Pickrell, A. M. & Youle, R. J. The roles of PINK1, parkin, and mitochondrial fidelity selective respiratory chain turnover in vivo. Proc. Natl Acad. Sci. USA 110, in Parkinson’sdisease. Neuron 85,257–273 (2015). 6400–6405 (2013). 4. Gustafsson,C.M., Falkenberg, M.&Larsson, N. G. Maintenance and 30. Baggio, F. et al. Drosophila melanogaster LRPPRC2 is involved in coordination expression of mammalian mitochondrial DNA. Annu. Rev. Biochem. 85, of mitochondrial translation. Nucleic Acids Res. 42,13920–13938 (2014). 133–160 (2016). 31. Szczepanowska, K. et al. CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels. EMBO J. 35,2566–2583 (2016). 5. Hallberg, B. M. &Larsson,N.G.Making proteins in the powerhouse. Cell Metab. 32. Camara, Y. et al. MTERF4 regulates translation by targeting the methyl- 20, 226–240 (2014). transferase NSUN4 to the mammalian mitochondrial ribosome. Cell Metab. 13, 6. Patron,M., Sprenger,H.G.&Langer,T.m-AAA proteases, mitochondrialcal- 527–539 (2011). cium homeostasis and neurodegeneration. Cell Res. 28, 296–306 (2018). Official journal of the Cell Death Differentiation Association Pareek et al. Cell Death Discovery (2019) 5:51 Page 14 of 14 33. Dogan,S.A.et al. Tissue-specific loss of DARS2 activates stress responses 49. Siegelin, M. D. et al. Exploiting the mitochondrial unfolded protein response independently of respiratory chain deficiency in the heart. Cell Metab. 19, for cancer therapy in mice and human cells. J. Clin. Invest. 121,1349–1360 458–469 (2014). (2011). 34. Bernstein, S. H. et al. The mitochondrial ATP-dependent Lon protease: a novel 50. Munch,C.& Harper,J.W.Mitochondrial unfolded proteinresponsecontrols target in lymphoma death mediated by the synthetic triterpenoid CDDO and matrix pre-RNA processing and translation. Nature 534,710–713 (2016). its derivatives. Blood 119,3321–3329 (2012). 51. Bratic, A. et al. The bicoid stability factor controls polyadenylation and 35. Suzuki, C. K., Suda, K., Wang, N. & Schatz, G. Requirement for the yeast gene expression of specific mitochondrial mRNAs in Drosophila melanogaster. PLoS LON in intramitochondrial proteolysis and maintenance of respiration. Science Genet. 7, e1002324 (2011). 264, 891 (1994). 52. Rackham, O. et al. Hierarchical RNA Processing Is Required for Mitochondrial 36. Bezawork-Geleta, A., Brodie,E.J., Dougan,D.A.&Truscott, K.N. LON is the Ribosome Assembly. Cell Rep. 16,1874–1890 (2016). master protease that protects against protein aggregation in human mito- 53. Bezawork-Geleta,A., Saiyed,T., Dougan, D. A.& Truscott, K.N. Mitochondrial chondria through direct degradation of misfolded proteins. Sci. Rep. 5, 17397 matrix proteostasis is linked to hereditary paraganglioma: LON-mediated (2015). turnover of the human flavinylation factor SDH5 is regulated by its interaction 37. Ron,D.& Walter,P.Signalintegration in the endoplasmic reticulum unfolded with SDHA. FASEB J. 28,1794–1804 (2014). protein response. Nat. Rev. Mol. Cell Biol. 8,519–529 (2007). 54. Matsushima, Y. et al. Drosophila protease ClpXP specifically degrades 38. Walter, P. & Ron, D. The unfolded protein response: from stress pathway to DmLRPPRC1 controlling mitochondrial mRNA and translation. Sci. Rep. 7,8315 homeostatic regulation. Science 334, 1081–1086 (2011). (2017). 39. Shpilka, T. & Haynes, C. M. The mitochondrial UPR: mechanisms, physiological 55. Siira, S. J. et al. LRPPRC-mediated folding of the mitochondrial transcriptome. functions and implications in ageing. Nat. Rev. Mol. Cell Biol. 19,109–120 Nat. Commun. 8,1532 (2017). (2018). 56. Gilkerson, R. et al. The mitochondrial nucleoid: integrating mitochondrial DNA 40. Pakos-Zebrucka, K. et al. The integrated stress response. EMBO Rep. 17, into cellular homeostasis. Cold Spring Harb. Perspect. Biol. 5, a011080 (2013). 1374–1395 (2016). 57. Gratz,S.J.etal. Highly specificand efficient CRISPR/Cas9-catalyzed homology- 41. Balch, W. E., Morimoto, R. I., Dillin, A. & Kelly, J. W. Adapting proteostasis for directed repair in Drosophila. Genetics 196,961–971 (2014). disease intervention. Science 319, 916–919 (2008). 58. Gargano,J.W., Martin,I., Bhandari,P.&Grotewiel, M. S. Rapiditerative negative 42. Lamech, L. T. & Haynes, C. M. The unpredictability of prolonged activation of geotaxis (RING): a new method for assessing age-related locomotor decline in stress response pathways. J. Cell Biol. 209, 781–787 (2015). Drosophila. Exp. Gerontol. 40,386–395 (2005). 43. Akerfelt, M., Morimoto, R. I. & Sistonen, L. Heat shock factors: integrators of 59. Greene, J. C. et al. Mitochondrial pathology and apoptotic muscle degen- cell stress, development and lifespan. Nat. Rev. Mol. Cell Biol. 11,545–555 eration in Drosophila parkin mutants. Proc. Natl Acad. Sci. USA 100,4078–4083 (2010). (2003). 44. Protter, D. S. & Parker, R. Principles and properties of stress granules. Trends Cell 60. Burman, J. L. et al. A Drosophila model of mitochondrial disease caused by a Biol. 26,668–679 (2016). complex I mutation that uncouples proton pumping from electron transfer. 45. Nargund, A. M., Fiorese, C. J., Pellegrino, M. W., Deng, P. & Haynes, C. M. Dis. Models Mech. 7, 1165–1174 (2014). Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 61. Tennessen,J.M., Barry,W.E., Cox, J. &Thummel, C. S. Methodsfor studying promotes OXPHOS recovery during the UPR(mt). Mol. Cell 58, 123–133 (2015). metabolism in Drosophila. Methods 68,105–115 (2014). 46. Nargund,A.M., Pellegrino, M. W.,Fiorese, C.J., Baker, B. M. &Haynes, C. M. 62. Jha,P., Wang,X.& Auwerx,J.Analysis ofmitochondrial respiratorychain Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR acti- supercomplexes using blue native polyacrylamide gel electrophoresis (BN- vation. Science 337,587–590 (2012). PAGE). Curr. Protoc. Mouse Biol. 6,1–14 (2016). 47. Zhao, Q. et al. A mitochondrial specific stress response in mammalian cells. 63. Pimenta de Castro, I. et al. Genetic analysis of mitochondrial protein misfolding EMBO J. 21, 4411–4419 (2002). in Drosophila melanogaster. Cell Death Differ. 19,1308–1316 (2012). 48. Owusu-Ansah, E., Song, W. & Perrimon, N. Muscle mitohormesis 64. Davis,M.Y.etal. Glucocerebrosidase deficiency in Drosophila results in alpha- promotes longevity via systemic repression of insulin signaling. Cell 155, synuclein-independent protein aggregation and neurodegeneration. PLoS 699–712 (2013). Genet. 12, e1005944 (2016). Official journal of the Cell Death Differentiation Association http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death Discovery Springer Journals

Lon protease inactivation in Drosophila causes unfolded protein stress and inhibition of mitochondrial translation

Download PDF
14 pages

Loading next page...
 
/lp/springer-journals/lon-protease-inactivation-in-drosophila-causes-unfolded-protein-stress-7iV31jamSc

References (68)

Publisher
Springer Journals
Copyright
Copyright © 2018 by The Author(s)
Subject
Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
eISSN
2058-7716
DOI
10.1038/s41420-018-0110-1
Publisher site
See Article on Publisher Site

Abstract

Mitochondrial dysfunction is a frequent participant in common diseases and a principal suspect in aging. To combat mitochondrial dysfunction, eukaryotes have evolved a large repertoire of quality control mechanisms. One such mechanism involves the selective degradation of damaged or misfolded mitochondrial proteins by mitochondrial resident proteases, including proteases of the ATPase Associated with diverse cellular Activities (AAA ) family. The importance of the AAA family of mitochondrial proteases is exemplified by the fact that mutations that impair their functions cause a variety of human diseases, yet our knowledge of the cellular responses to their inactivation is limited. To address this matter, we created and characterized flies with complete or partial inactivation of the Drosophila matrix-localized AAA protease Lon. We found that a Lon null allele confers early larval lethality and that severely reducing Lon expression using RNAi results in shortened lifespan, locomotor impairment, and respiratory defects specific to respiratory chain complexes that contain mitochondrially encoded subunits. The respiratory chain KD defects of Lon knockdown (Lon ) flies appeared to result from severely reduced translation of mitochondrially encoded genes. This translational defect was not a consequence of reduced mitochondrial transcription, as evidenced KD by the fact that mitochondrial transcripts were elevated in abundance in Lon flies. Rather, the translational defect of KD Lon flies appeared to be derived from sequestration of mitochondrially encoded transcripts in highly dense KD ribonucleoparticles. The translational defect of Lon flies was also accompanied by a substantial increase in unfolded mitochondrial proteins. Together, our findings suggest that the accumulation of unfolded mitochondrial proteins triggers a stress response that culminates in the inhibition of mitochondrial translation. Our work provides a foundation to explore the underlying molecular mechanisms. Introduction stoichiometric imbalance between mitochondrial and Mitochondria are responsible for most of the energy nuclear encoded respiratory chain subunits can cause produced by a cell, but the generation of reactive oxygen misfolding and aggregation of the unassembled pro- 4,5 species (ROS) as a byproduct of this activity can damage teins . Fortunately, there are many surveillance pathways 1–3 + mitochondrial proteins, lipids, and DNA . Also, while that oppose or reverse this damage, including the AAA 6–8 + mitochondria contain their own genome, most mito- family of mitochondrial proteases . All of the AAA chondrial proteins are encoded in the nucleus, and a proteases form multimeric protein complexes and use ATP to unfold and transport substrates to an internal proteolytic cavity for degradation. In higher eukaryotes, there are five major mitochondrial AAA proteases that Correspondence: Leo J. Pallanck (pallanck@uw.edu) Department of Genome Sciences, University of Washington, 3720 15th are distinguished by their subunit composition and Avenue NE, Seattle, WA 98195, USA mitochondrial localization. The most well-studied mem- Department of Biochemistry, University of Washington, 1705 NE Pacific St., ber of the AAA protease family is Lon. Seattle, WA 98195, USA Edited by I. Amelio © 2018 The Author(s) Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Pareek et al. Cell Death Discovery (2019) 5:51 Page 2 of 14 Previous work indicates that Lon possesses three dif- an unfolded protein stress response that inhibits mito- ferent activities, serving as a chaperone and DNA-binding chondrial translation. 9,10 protein in addition to its role in proteolysis . Lon expression is regulated by multiple cellular stresses, Results including ROS and unfolded protein stress, and there is Creation of Lon-deficient Drosophila strains substantial support for the role of Lon in the degradation To explore the biological roles of Lon protease we used 11,12 of oxidatively damaged and misfolded proteins . CRISPR/Cas9 technology to create a null allele of the Although few Lon substrates are known with certainty, a Drosophila Lon gene (CG8798). Briefly, we constructed number of candidate Lon substrates have been identified guide RNAs designed to create double-strand breaks from biochemical studies aimed at the identification of flanking the Lon coding sequence (Suppl. Figure 1). We Lon-binding proteins, including the mitochondrial DNA also created a donor vector construct consisting of the (mtDNA) replication factors Twinkle, polymerase gamma, DsRed marker flanked by 5′ and 3′ untranslated sequences 13–19 Tfam, and the chaperones Hsp60 and mtHsp70 . from the Lon coding region for use in homology-directed Subsequent studies aimed at validating the significance of recombinational repair of the double-strand breaks. Flies these binding interactions indicated that Lon inactivation expressing the DsRed marker were then subjected to results in increased mtDNA copy number and destabili- whole-genome sequencing to verify correct targeting of zation of Hsp60 and mtHsp70 under environmental DsRed to the Lon locus and complete deletion of the Lon 20–22 stress . However, it is unclear whether these findings gene. Flies heterozygous for this deletion were fully viable reflect direct effects of Lon inactivation, or downstream with no detectable phenotypes. However, homozygotes cellular responses to loss of Lon activity. Moreover, died at the second instar larval stage of development, mutations in Lon have been shown to result in the demonstrating that Lon is essential for viability (Suppl. recessive developmental disorder CODAS (cerebral, Figure 2a). ocular, dental, auricular, and skeletal) syndrome, yet the RNAi often reduces but does not completely eliminate mechanisms by which mutations in Lon cause this disease target gene expression. Thus, we tested whether RNAi are currently unknown . lines targeting Lon would circumvent the lethality con- To create a simple, genetically tractable model system ferred by a null mutation in Lon. We tested two different to explore the biological role of Lon and the pathological RNAi constructs targeting Lon (designated Lon-RNAi-1 consequences of Lon inactivation, we used CRISPR/Cas9- and Lon-RNAi-2) that we used in previously published mediated gene targeting and RNAi to create Drosophila work to explore the influence of Lon on the abundance strains with complete and partial loss of Lon function. We and activity of the mitophagy factor PINK1 . Our pre- found that Lon is an essential gene in Drosophila and that vious work demonstrated that these RNAi lines differed in flies expressing RNAi against Lon (Lon knockdown flies, the efficiency by which they reduced Lon expression, with KD or Lon flies) had shortened lifespan, defective locomo- the Lon-RNAi-2 line resulting in greater reduction in Lon tion, and altered respiratory chain activity. The respiratory expression. We expressed each RNAi line using two GAL4 KD chain deficits in Lon flies were specific to respiratory drivers simultaneously: the pan-neuronal elav-GAL4 dri- chain complexes that contain subunits encoded by the ver and the ubiquitous da-GAL4 driver. Driving the mitochondrial genome, suggesting that altered expression stronger Lon-RNAi-2 line with this combination of GAL4 of mitochondrially encoded components underlies this drivers failed to yield viable adult flies, but flies expressing defect. Consistent with this conclusion, we found that the weaker Lon-RNAi-1 transgene were fully viable and reduced mitochondrial complex activity is accompanied fertile as young adults, and had no obvious morphological by reduced complex abundance and diminished mito- alterations. Western blot analyses performed on heads KD chondrial translation. The translational defect of Lon and whole flies indicated that Lon expression was sig- flies was not a consequence of reduced mitochondrial nificantly reduced in flies bearing the UAS-Lon-RNAi-1, transcription, but rather appeared to be a consequence of elav-GAL4, and da-GAL4 transgenes compared to con- reduced association of mitochondrial transcripts on trols expressing RNAi against the exogenous mCherry mitochondrial ribosomes and packaging of mitochondrial sequence (UAS-mCherry-RNAi) (Fig. 1a and Suppl. Fig- transcripts into highly dense ribonucleoparticles. The ure 2b). Flies bearing the Lon-RNAi-1, elav-GAL4, and da- KD translational defect of Lon flies was also accompanied GAL4 transgenes were used in most of the remaining KD by elevated abundance of unfolded mitochondrial pro- studies and will hereafter be called Lon . teins, and overexpression of another matrix protease, ClpP, partially rescued the defects associated with Lon Partial inactivation of Lon results in shortened lifespan, inactivation, possibly by reducing the burden of unfolded locomotor defects, and altered respiratory chain activity KD mitochondrial proteins. Together, our findings strongly To further characterize the Lon phenotypes, we suggest that Lon inactivation results in the activation of subjected the flies to several standard Drosophila Official journal of the Cell Death Differentiation Association Pareek et al. Cell Death Discovery (2019) 5:51 Page 3 of 14 a b c d kDa 8 1.0 ** α Lon *** 0.8 α Actin 0.6 0.4 1.2 *** 1.0 2 0.2 0.8 0.6 Days 0.4 0.2 Fig. 1 Inactivation of Lon results in shortened lifespan and defective locomotion. a Immunoblot analysis from heads of 1-day-old control and KD Lon flies using Lon and actin antibodies. The Lon band intensity is normalized against actin as a loading control. Significance was determined using KD Student’s t-test (***p< 0.0005 by Student’s t-test). The experiment was repeated at least three times. b Kaplan–Meier survival curves of Lon flies KD (n = 167, 50% survival 31 days) and controls (n = 174, 50% survival 73 days) (****p < 0.0001 by Mantel–Cox log-rank test). c One-day-old Lon flies KD KD exhibit a climbing defect. Error bars represent SEM (n = 135 for Lon , 134 for control, ***p < 0.0005 by Student’s t-test). d One-day-old Lon flies exhibit a significant decrease in flight index (n = 6 independent groups of 10–15 animals, **p < 0.005 from Student’s t-test). Control= UAS-mCherry- KD RNAi driven by elav-GAL4 and da-GAL4. Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 KD behavioral assays. Although Lon flies appeared normal Figure 3). Together, our findings indicate that partial upon eclosion, they were significantly shorter-lived than inactivation of Lon results in a progressive age-dependent the control flies (Fig. 1b). The maximal and median life- decline in oxidative phosphorylation capacity, and that KD spans of Lon flies were similar to those of flies bearing this decline may underlie the shortened lifespan and + KD null mutations in genes encoding the other AAA mito- behavioral deficits of Lon flies. chondrial protease family members dYME1L and 25,26 KD SPG7 . Young Lon flies also exhibited defects in Lon knockdown results in increased mtDNA-encoded climbing and flight starting early in life (Fig. 1c, d). transcript abundance and reduced translation Inactivation of Lon in other model systems results in All of the respiratory chain complexes exhibiting 27 KD reduced respiratory chain activity . Thus, we tested reduced abundance and assembly in Lon flies (com- KD whether Lon flies exhibit similar respiratory chain plexes I, III, IV, and V) contain one or more subunits defects, using established biochemical assays to quantify encoded by mtDNA. Additionally, our BN-PAGE and in- KD respiratory chain activity in young (1 day) and old gel activity assays in Lon flies indicated that the ATP KD (3 weeks) Lon flies. We found that respiratory chain synthase F subunit containing subcomplex (consisting KD complexes I and IV had reduced activity in old Lon flies entirely of subunits encoded by nuclear DNA) accumu- compared to the controls, while young flies showed lated at the expense of the F subcomplex (which includes reduction only in complex I activity (Fig. 2a). However, two subunits encoded by mtDNA). By contrast, complex KD complex II activity was increased significantly in both II, which exhibited increased activity in Lon flies, con- KD young and old Lon flies. These alterations could result sists entirely of nuclear DNA–encoded components. from a functional change in respiratory chain activity or These findings led us to hypothesize that reduced mtDNA from altered respiratory chain abundance. To distinguish abundance, reduced transcription of mtDNA-encoded between these possibilities while simultaneously con- subunits, and/or reduced translation of mitochondrial firming and extending our biochemical studies, we transcripts might account for the decreased expression of assessed the abundance of assembled complexes and their complexes I, III, IV, and V. corresponding activities using blue native PAGE (BN- To begin to explore these hypotheses, we first compared 28 KD PAGE) and in-gel enzyme activity assays . This work mtDNA abundance in Lon flies and the controls. While revealed that complexes I, III, and IV all exhibited both increased mtDNA abundance in response to Lon inacti- KD 20 reduced activity and reduced abundance in Lon flies, vation has been reported , mtDNA copy number was KD whereas complex II exhibited an increase in activity unchanged in Lon flies (Fig. 3a). This is consistent with (Fig. 2b–d). Additionally, this work revealed the presence our previous finding of unchanged mtDNA abundance of a partially assembled but catalytically active F sub- when Lon was knocked down with elav-GAL4 alone . unit–containing subcomplex of ATP synthase (complex We next analyzed the steady-state levels of several mito- V) (Fig. 2b, e). These alterations were also accompanied chondrial transcripts by qRT-PCR. We found that levels KD by a reduction in ATP content in Lon flies (Suppl. of all transcripts analyzed, including 12S and 16S rRNA Official journal of the Cell Death Differentiation Association Fold Change Percent Survival Distance Climbed (cm) Flight Index Pareek et al. Cell Death Discovery (2019) 5:51 Page 4 of 14 Complex IV 2.0 1.0 Day 1 Day 21 Complex IV ns 0.8 1.5 0.6 Complex III Complex III ns 1.0 ns Complex I Complex II Complex II Complex I 0.4 ** ** ** 0.5 0.2 0 0 b c d e kDa kDa kDa kDa C(V) SCs 2 C(V) CI CV CI 720 C(V) CIII 720 720 * C(IV) /CIII CIV CII 242 CIV CII 146 146 66 66 kDa Citrate synthase Fig. 2 Lon knockdown flies have altered respiratory chain function and abundance. a The activity of respiratory chain complexes in control KD and Lon flies at 1 day (left panel) and 21 days (right panel) of age (n = 2 independent groups of 500 adult flies, *p < 0.05, **p < 0.005 from Student’s KD t-test). b BN-PAGE analysis of mitochondrial protein extracts from 21-day-old control and Lon flies. The red asterisk marks the location of KD the subcomplex containing F subunit of ATP synthase that is only detected in Lon flies. Immunoblot of citrate synthase (bottom panel) was used as a loading control. c In-gel activity of mitochondrial complexes I and IV isolated from 21-day-old adults. SCs here refer to the supercomplexes. d In- gel activity of mitochondrial complex II isolated from 21-day-old flies. e In-gel activity of mitochondrial complex V isolated from 21-day-old adults. KD The red asterisk denotes the location of the subcomplex containing F subunit of ATP synthase in Lon flies. Note that the protein molecular weight markers shown in (b) were used as reference to mark the gels in (c–e). For BN-PAGE and in-gel activity assays, the images shown are representative of KD two independent biological replicates. Control = UAS-mCherry-RNAi driven by elav-GAL4 and da-GAL4. Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 KD and several mRNAs, were increased in old Lon flies This finding may reflect the fact that mitochondrial pro- (Fig. 3b). Additionally, the transcript abundance of the teins are extremely long-lived and that there is thus a mitochondrial transcription-promoting factors TFAM decreased need for mitochondrial translation during the KD and mtTFB2 was unchanged in Lon flies (Suppl. Fig- adult stage of development. However, previous work has ure 4). Together, these findings indicate that the respira- detected robust labeling of mitochondrial proteins using KD tory chain defects in Lon flies do not derive from mitochondria obtained from larvae, so we repeated our in reduced mitochondrial gene dosage or reduced mito- organello labeling studies using mitochondria from third chondrial transcription. instar larvae . Our western blot analysis confirmed that KD KD To test whether Lon flies manifest a translational Lon expression was greatly reduced in Lon larvae defect, we used S-labeled methionine to perform in relative to the controls (Fig. 3c). This experiment revealed organello labeling of mitochondria prepared from adult a substantial decrease in de novo labeling of mitochon- KD flies. However, we detected very little labeling of mito- drial translation products in Lon larvae relative to the chondrial proteins in our control flies (Suppl. Figure 5). controls (Fig. 3d), suggesting that the decreased Official journal of the Cell Death Differentiation Association Normalized respiratory chain activity Pareek et al. Cell Death Discovery (2019) 5:51 Page 5 of 14 a b ns 1.0 ** ** *** ** 0.5 ** ** kDa kDa 96 Lon Actin ** 1.0 COX1 CYTB 0.5 COXIII COXII/ATP6 ND4L/ ATP8 Fig. 3 Lon knockdown flies exhibit increased mitochondrial transcript abundance and decreased mitochondrial translation. a Mitochondrial KD DNA abundance was compared in Lon and control flies using qPCR to compare the ratio of mtDNA-encoded mt:Cyt-b to that of nuclear-encoded Act79b (n = 3 independent groups of 40–45 fly heads). Statistical significance was determined using Student’s t-test. b qRT-PCR was used to quantify steady-state abundance of the indicated mitochondrial RNAs in 21-day-old adult fly heads. Mitochondrial RNA abundance was normalized to the abundance of the nuclear-encoded Act79b transcript (n = 3 independent groups of 40–45 fly heads). Error bars indicate mean ± SEM. Student’s t-test was applied, *p < 0.05, **p < 0.005, ***p < 0.0005. c Immunoblot analysis of third instar larvae to confirm knockdown of Lon using actin as a loading control. n = 3 independent groups of five third instar larvae. Significance was determined using Student’s t-test, **p < 0.005. d In organello translation was performed using mitochondria isolated from third instar larvae. Mitochondria were labeled by incubating with S-methionine for 1 h. Positions of individual mitochondrially encoded proteins are indicated (left panel). Coomassie-stained gel (right panel) was used as a loading control. KD Control= UAS-mCherry-RNAi driven by elav-GAL4 and da-GAL4. Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 KD abundance of complexes I, III, IV, and V in Lon flies is a Mitochondrial transcripts are packaged into untranslated KD direct consequence of a translational defect. The particles in Lon flies increased abundance of mtDNA-encoded transcripts and To investigate the mechanism by which inactivation of nuclear DNA–encoded complex II may represent com- Lon impairs mitochondrial translation, we assessed the pensatory responses to alleviate this translational defi- state of mitochondrial ribosome assembly by performing 31–33 ciency . sucrose density gradient sedimentation analyses. Official journal of the Cell Death Differentiation Association mtDNA/NucDNA ratio Fold Change KD Log Fold Change (Lon /control) 2 Pareek et al. Cell Death Discovery (2019) 5:51 Page 6 of 14 gradient were then subjected to qRT-PCR to quantify the distribution of the 12S and 16S mitochondrial ribosomal 12S control RNAs, which mark the small (28S) and large (39S) KD Lon 28S 39S 55S mitochondrial ribosomal subunits, respectively. Co- localization of the 12S and 16S mitochondrial ribosomal RNAs within the same fraction is diagnostic for fully assembled and actively translating mitochondrial ribo- 20 somes (55S). This analysis revealed that the abundance of 12S and 16S ribosomal RNAs in fully assembled mito- chondrial ribosome fractions (fraction 15–17) was KD decreased by 5.8% and 6.4%, respectively, in Lon flies relative to the controls (Fig. 4). The mild decrease in mitochondrial ribosome assembly appeared insufficient to account for the translational 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 KD defect of Lon flies, especially in light of the fact that Fraction number mitochondrial transcripts are elevated in abundance relative to the controls. Thus, we examined other possible explanations for the translational defect of Lon-deficient 16S flies. One possible explanation of our findings was that control mitochondrial transcripts are translated at lower effi- KD KD 28S 39S 55S Lon ciency in Lon flies. To test this model, we used sucrose density gradient centrifugation to quantify the relative distribution of four different mitochondrial transcripts, including mt:ND5, mt:Cyt-b, mt:CoII, and ATPase8/6,in gradient fractions (Fig. 5). All of these mRNAs displayed a predominant sedimentation peak co-migrating with the KD 55S ribosome in control samples. By contrast, in Lon flies a smaller proportion of these transcripts co-migrated with the 55S ribosome, and this decrease was accom- panied by a striking increase in the proportion of these transcripts that sedimented to the bottom of the gradient 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 (Fig. 5). This finding suggests that a substantial proportion Fraction number KD of the mitochondrial transcripts in Lon flies are pack- aged into large untranslated particles. Fig. 4 Mitochondrial ribosome assembly is only mildly affected in KD KD Lon flies. Mitochondrial lysates from 21-day-old control and Lon Lon knockdown flies accumulate unfolded mitochondrial flies were subjected to sucrose density gradient fractionation to assess the state of assembly of mitochondrial ribosomes. The relative proteins and trigger the mitochondrial unfolded protein proportions of the small (28S) subunit, large (39S) subunit, and fully response assembled (55S) mitochondrial ribosomes were assessed by Lon is believed to play a critical role in the degradation subjecting the density gradient fractions to qRT-PCR using primer sets 34–36 of unfolded mitochondrial proteins . The accumula- specificto 12S rRNA, which marks the small subunit, and to 16S rRNA, tion of unfolded proteins in multiple cellular compart- which marks the large subunit. Co-localization of the 12S and 16S rRNAs is diagnostic of fully assembled and actively translating ments has been shown to activate a stress response known ribosomes. Fractions containing the small (28S) subunit, large as the unfolded protein response (UPR) that is specific (39S) subunit, and fully assembled (55S) ribosome are shaded cyan, to the compartment in which the unfolded proteins pink, and yellow, respectively. The relative abundance of a given rRNA 37–39 reside . The cytosolic and endoplasmic reticulum (ER) in each fraction was calculated as the percentage relative to the total UPR restore protein homeostasis through a two-tiered RNA abundance in all fractions after normalizing to a luciferase control RNA that was spiked into each of the fractions prior to RNA isolation. system consisting of increased expression of chaperones Control= UAS-mCherry-RNAi driven by elav-GAL4 and da-GAL4. to facilitate the refolding of misfolded proteins, and KD Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 phosphorylation-mediated inactivation of the cytosolic translation-initiation factor eIF2α to attenuate transla- tion . While previous work has established that unfolded KD Mitochondrial extracts from Lon flies and controls protein stress in the mitochondria triggers the induction were subjected to sucrose density gradient analysis as of chaperones and proteases, whether mitochondrial previously described . Fractions from the sucrose translation is inhibited by unfolded protein stress is less Official journal of the Cell Death Differentiation Association Relative abundance Relative abundance Pareek et al. Cell Death Discovery (2019) 5:51 Page 7 of 14 mt:ND5 ATPase8/6 control control KD KD Lon 28S 39S 55S 28S 39S 55S Lon 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Fraction number Fraction number control control mt:CoII mt:Cyt-b KD KD Lon Lon 28S 39S 55S 28S 39S 55S 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Fraction number Fraction number Fig. 5 Mitochondrial RNAs accumulate in large untranslated particles upon Lon knockdown. qRT-PCR analysis of individual sucrose gradient fractions was used to characterize the distribution of the indicated mtDNA-encoded mRNAs relative to the 28S and 39S subunits, and fully KD assembled 55S mitochondrial ribosomes. Mitochondrial homogenates for sucrose density fractionation were prepared from 21-day-old adult Lon and control flies. Relative abundance represents the fraction of the mRNA in any given fraction relative to the total after normalizing to a luciferase KD control RNA that was added to each of the fractions prior to RNA isolation. Control= UAS-mCherry-RNAi driven by elav-GAL4 and da-GAL4. Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 clear. Our findings that Lon inactivation appears to result to selected mitochondrial proteins to quantify the pro- in the sequestration of mitochondrially encoded mRNAs portion of each protein in the soluble and insoluble KD into translationally inactive particles, coupled with the fractions. Lon flies had higher levels of insoluble severe defect in mitochondrial translation, led us to mitochondrial proteins than the control flies at both day 1 hypothesize that the mitochondrial UPR triggers transla- and day 21 of age (Fig. 6a and Suppl. Figure 6a), indicating KD tional inhibition. that Lon flies accumulate unfolded mitochondrial pro- To explore our hypothesis we first analyzed whether teins. We next examined the abundance of the mito- KD KD Lon flies accumulate unfolded mitochondrial proteins chondrial UPR markers Hsp60 and Hsc70-5 in Lon and whether they activate the mitochondrial UPR. To test flies. This analysis revealed a marked increase in both KD whether unfolded mitochondrial proteins accumulate in Hsp60 and Hsc70-5 in Lon flies at day 1 and at day 21 KD Lon flies, we prepared fly head protein extracts using of age (Fig. 6b and Suppl. Figure 6b). Together, these KD Triton X-100. We then subjected Triton-soluble and findings indicate that Lon flies accumulate unfolded -insoluble proteins to western blot analysis using antisera proteins and trigger the mitochondrial UPR. Official journal of the Cell Death Differentiation Association Relative abundance Relative abundance Relative abundance Relative abundance Pareek et al. Cell Death Discovery (2019) 5:51 Page 8 of 14 4.5 kDa *** 3.5 ** 2.5 1.5 0.5 KD Lon aconitase compV PDH NDUFS3 3.5 ** 2.5 kDa 1.5 0.5 KD Lon Hsp60 Hsc70-5 Fig. 6 Inactivation of Lon protease results in the accumulation of unfolded mitochondrial proteins. a Triton-insoluble mitochondrial proteins KD detected by western blot in heads from 1-day-old control and Lon flies, using antibodies to complex Vβ, PDHα, aconitase, and NDUFS3. The results were quantified by ratio to actin and normalized to control levels. The experiment was repeated at least three times. *p< 0.05, **p< 0.005, ***p< 0.0005 by Student’s t-test. b Immunoblot analysis of head proteins using antibodies to Hsp60A and Hsc70-5. Protein was extracted from heads of day KD 1 control and Lon flies using RIPA buffer. Quantification was performed as in (a). Experiments were repeated at least three times. *p< 0.05, **p< KD 0.005 by Student’s t-test. Control = UAS-mCherry-RNAi driven by elav-GAL4 and da-GAL4. Lon = UAS-Lon-RNAi-1 driven by elav-GAL4 and da-GAL4 Overexpression of ClpP protease ameliorates the defect of compared Lon knockdown flies overexpressing ClpP to Lon knockdown flies Lon knockdown flies expressing mCherry RNAi. How- KD Protein quality control in the mitochondrial matrix is ever, strong overexpression of ClpP in Lon flies wor- regulated by an elaborate network of proteases and cha- sened the locomotor defect (Suppl. Figure 7). Thus, we perones, including the AAA protease family member tested whether ClpP expression could rescue a climbing Clp protease. To test whether the accumulation of defect caused by expressing the Lon-RNAi-1 transgene KD KD-elav unfolded proteins in Lon flies is responsible for the using just the elav-GAL4 driver (designated as Lon ). phenotypes associated with Lon inactivation, we co- We first confirmed that driving the UAS-ClpP transgene expressed the proteolytic subunit of the Drosophila Clp with elav-GAL4 resulted in the production of detectable protease (CG5045, hereafter ClpP) along with the Lon- ClpP protein (Fig. 7a) and use of the elav-GAL4 driver in RNAi-1 RNAi construct and examined whether ClpP conjunction with the Lon-RNAi-1 transgene reduced Lon overexpression is capable of rescuing the locomotor expression (Suppl. Figure 8) and caused a climbing defect KD defect of Lon flies. To account for possible titration of (Fig. 7b). Results of this analysis revealed that ClpP GAL4 protein in the presence of two UAS transgenes, we overexpression rescued the locomotor defect of Lon Official journal of the Cell Death Differentiation Association Fold Change Insoluble Fraction Fold Change Pareek et al. Cell Death Discovery (2019) 5:51 Page 9 of 14 orders of magnitude greater than that of the nuclear genome . Moreover, expression of the mitochondrial and nuclear genomes must be coordinated to ensure proper stoichiometry of mitochondrial and nuclear-encoded kDa respiratory chain components. An imbalance in this coordination can result in the accumulation of misfolded 39 + proteins and mitochondrial dysfunction . The AAA Flag mitochondrial protease family is believed to play an important role in maintaining mitochondrial competence by degrading and thereby facilitating the replacement of Actin 43 oxidatively damaged and misfolded proteins. However, we know little of the cellular responses and the pathogenic mechanisms of diseases caused by mutations in the genes that encode the AAA proteases. Our current work advances our understanding of these matters by showing *** that inactivation of the AAA protease Lon results in **** respiratory chain defects that appear to result from translational inhibition. Our findings further indicate that the translational defect caused by Lon inactivation results at least in part from the sequestration of mtDNA-encoded mRNAs into translationally inactive particles. Finally, we find that insoluble mitochondrial matrix proteins accu- KD mulate in Lon animals, suggesting that mitochondrial protein aggregation triggers translational inactivation as a stress response. Our work provides a foundation to fur- ther explore the mechanisms by which mitochondrial unfolded protein stress inhibits mitochondrial translation. Protein unfolding in the ER and cytoplasm induce well- 37,38,40–43 characterized stress responses . Each of these stress pathways has two arms: increased expression of chaperones to refold proteins, and translational inhibition to limit further protein synthesis. Translational inhibition is mediated by phosphorylation of eIF2α and culminates in the formation of translationally stalled ribonucleopro- Fig. 7 ClpP overexpression rescues the climbing defect of Lon- tein/mRNA complexes known as stress granules . More deficient flies. a Immunoblot analysis of fly heads to confirm recently, work in a variety of experimental systems has expression of FLAG-tagged ClpP in flies bearing a UAS-ClpP-FLAG-HA established the existence of a mitochondria-specific ver- transgene and the elav-GAL4 pan-neuronal driver. b Climbing was mt 45–49 KD-elav sion of the unfolded protein response (UPR ) . measured in 1-day-old control flies (n = 94), Lon flies (n = 91), KD-elav Although the degree to which this pathway is evolutio- and Lon flies co-expressing ClpP protease (n = 83). Control = KD-elav UAS-mCherry-RNAi driven by elav-GAL4. Lon ; mCherry RNAi = UAS- narily conserved remains an active area of investigation, it KD-elav Lon-RNAi-1 and UAS-mCherry-RNAi driven by elav-GAL4. Lon ; UAS- has been shown in both vertebrate and invertebrate ClpP= UAS-Lon-RNAi-1 and UAS-ClpP-FLAG-HA driven by elav-GAL4. models that mitochondrial unfolded protein stress trig- Error bars represent SEM. ***p< 0.0005, ****p< 0.0001 by Student’s gers the transcriptional activation of genes involved in t-test 45,47 protein folding and degradation . However, previous mt work has not clearly established whether the UPR also knockdown flies, thus supporting the hypothesis that the results in inhibition of mitochondrial translation. Recently accumulation of unfolded proteins is responsible for the published findings in vertebrate cell culture have revealed phenotypes of Lon-deficient flies (Fig. 7b). that inactivation of the matrix chaperone Trap1 results in inhibition of mitochondrial translation as a consequence Discussion of a pre-RNA processing defect . Our findings that Lon Mitochondria are particularly prone to damage. They knockdown flies have both an excess of mitochondrial are the primary source and recipient of damaging ROS unfolded proteins and an impairment of mitochondrial mt and continual replication of the mitochondrial genome translation raise the possibility that the UPR , like other throughout life culminates in mutation frequencies often unfolded protein stress pathways, has a second arm Official journal of the Cell Death Differentiation Association Distance Climbed (cm) Pareek et al. Cell Death Discovery (2019) 5:51 Page 10 of 14 involving translational inhibition. However, the precise Materials and methods mechanism by which mitochondrial unfolded protein Fly stocks and maintenance stress in Lon-deficient animals inhibits mitochondrial Drosophila stocks were maintained on cornmeal- translation will require further investigation. molasses food at 25 °C on a 12 h:12 h light–dark cycle. Although most of the respiratory chain complexes The UAS-Lon-RNAi-1 construct P{GD14030}-v36036 was exhibited reduced activity and abundance upon Lon obtained from the Vienna Drosophila Resource Center. inactivation, complex II activity was increased. This The w , UAS-mCherry RNAi (P{VALIUM20-mCherry} increase may simply represent a compensatory response, attP2), UAS-Lon-RNAi-2 (P{TRiP.HMS01060}attP2), as complex II has no mitochondrially encoded subunits, elav-GAL4, and da-GAL4 driver lines were obtained from and increased complex II activity has previously been the Bloomington Stock Center (Bloomington, IN, USA). observed in mutants with defects in mitochondrial gene The UAS-ClpP-FLAG-HA transgenic line was obtained 30,51,52 expression . However, it is also possible that from the Fly Facility, National Centre for Biological Sci- increased complex II activity is a consequence of reduced ences, Bangalore, India. degradation of the complex II assembly factor Sdh5, given The Lon knockout allele was created using CRISPR/ that previous work has shown that Lon mediates the Cas9-mediated gene editing according to a published 53 25,57 turnover of Sdh5 . Lon knockdown might thus reduce procedure . Briefly, we replaced the Lon (CG8798) the degradation of Sdh5 and promote complex II assem- coding sequence with DsRed through homology-mediated bly. The increased abundance of mitochondrially encoded repair. The following primer sequences were used for transcripts upon Lon inactivation may likewise reflect guide RNAs targeting the 5′ and 3′ UTR regions of Lon: either a general compensatory response or a specific 5′-Guide RNA change due to altered turnover of a possible Lon sub- Sense oligo: 5′-CTTCGATAATCACTCACCACACAT strate. Increased mitochondrial transcript abundance has T-3′ been reported previously in mutants with defective Antisense oligo: 5′-AAACAATGTGTGGTGAGTGAT mitochondrial RNA processing and translation, suggest- TATC-3′ ing that increasing the abundance of mitochondrial 3′-Guide RNA mRNA is a common response to inadequate mitochon- Sense oligo: 5′-CTTCGGGGTGTTGCGGGTGTTGA 30,31,52 drial translation . However, the accumulation of T-3′ KD mitochondrially encoded RNAs in Lon flies could also Antisense oligo: 5′-AAACATCAACACCCGCAACAC CCC-3′ be explained by the finding that these flies accumulate the RNA-binding protein Lrpprc1 (leucine-rich penta- Sequences flanking the Lon coding region were ampli- tricopeptide repeat motif-containing 1 protein; Suppl. fied from genomic DNA to facilitate homology-directed Figure 9), which is known to stabilize mitochondrial repair using the following primer sequences: 51,54,55 transcripts . Finally, Lon is a known component of 5′-Homology arm mitochondrial nucleoids, and could therefore influence Forward: 5′-CCGGCACCTGCGGCCTCGCAGTGCT the turnover and abundance of other proteins involved in CCGATCACGTTGGGAATGGG-3′ the synthesis or stabilization of mitochondrial tran- Reverse: 5′-CCGGCACCTGCGGCCCTACGTGTGGT scripts . Future work will be required to address these GAGTGATTATGTGACGGCTGGTG-3′ matters. 3′-Homology arm There are many unanswered questions regarding the Forward: 5′-GGCCGCTCTTCATATGATAGGTTTTA biological roles of the AAA protease family and the TAAATATCTATCGTTATCAGG-3′ mitochondrial response to unfolded protein stress. For Reverse: 5′-CCGGGCTCTTCTGACTTTCCCCGCCT example, mutations in the human gene encoding Lon CACCGGTGGACGGCC-3′ cause a developmental disorder known as CODAS syn- Thesehomologyarmswerethenclonedintothe pHD- drome . However, the underlying mechanisms causing DsRed-attP vector containing the eye-specific 3xP3 disease are completely unknown. Although Lon is well promoter fused with DsRed and the resulting construct known to promote the degradation of oxidatively was microinjected into Cas9-expressing embryos by a damaged and unfolded proteins, the specific protein commercial service (Rainbow Transgenic Flies Inc.). substrates of Lon are largely unknown. Whether unfolded Flies bearing the Lon deletion were identified by protein stress in the mitochondria generally triggers screening the offspring of injected adults for expression translation inhibition, or this feature is specific to Lon of red fluorescence in the compound eye. Flies expres- inactivation is also unclear. Our study provides a foun- sing the DsRed marker were then further subjected to dation to address these questions and the mechanisms whole-genome sequencing to verify deletion of the Lon underlying CODAS syndrome in future work. gene. Official journal of the Cell Death Differentiation Association Pareek et al. Cell Death Discovery (2019) 5:51 Page 11 of 14 Lifespan and behavioral analyses the oxidation of NADH at 340 nm using ubiquinone-1 Lifespan and behavioral analyses were performed using as an electron acceptor. Nonspecific activity was deter- male flies. Age in all these experiments refers to the mined using 10 μM rotenone and subtracted to calculate number of days following eclosion. Longevity assays were complex I-specific activity. Complex II activity was performed at 25 °C and involved 20 flies per vial. Flies determined by monitoring the reduction of 2,6-dichlor- were transferred to fresh food every 2–3 days and the ophenolindophenol at 600 nm in the presence of succi- nate and decylubiquinone. Background activity was number of dead flies was recorded during each transfer. Kaplan–Meier lifespan curves were generated using determined using 10 mM malonate and subtracted to GraphPad Prism v5, and we used the Mantel–Cox log- calculate complex II-specific activity. Complex III activity rank test to determine the statistical significance of dif- was determined by monitoring the reduction of cyto- ferences in survival between tested genotypes. chrome c at 550 nm in a reaction mixture containing For climbing and flight assays, flies were anesthetized decylubiquinol and cytochrome c. Nonspecific activity with CO and allowed to recover for at least 24 h before was determined using antimycin A and subtracted to the experiment. Climbing behavior was assessed using the calculate complex III-specific activity. Complex IV activity Rapid Iterative Negative Geotaxis (RING) assay at day 1 assay was performed by monitoring the oxidation of according to a previously published protocol with minor reduced cytochrome c at 550 nm. Background activity was modifications . In particular, height climbed was mea- determined using potassium cyanide and used to calculate sured on still images from video recordings rather than on complex IV-specific activity. All activities were normal- photographs. Briefly, 15 flies were transferred into plastic ized to citrate synthase activity, which was determined by vials and these vials were then loaded onto the RING following the reduction of 5,5′-dithiobis (2-nitrobenzoic apparatus. The apparatus was tapped down to initiate the acid) at 412 nm in the presence of acetyl-coenzyme A and climbing response and the height climbed by each fly after oxaloacetate. 3 s was recorded. The climbing assay was repeated three times for each group. Total ATP determination Flight assays were performed according to a previously Total ATP was determined from whole flies according 59,60 61 published protocol . Briefly, an acetate sheet was to a previously published procedure . Briefly, five 21-day- divided into five equal parts, coated with grease and old flies were homogenized in 100 μL of homogenization inserted into a 2-liter graduated cylinder. One-day-old buffer (6 M guanidine HCL, 100 mM Tris (pH 7.8), and 4 flies were tapped into a funnel at the top of the cylinder mM EDTA). The samples were boiled for 5 min and and became stuck to the vacuum grease where they subjected to centrifugation for 3 min at 21,000 g to alighted. The number of flies that alighted in each of the remove debris. The supernatant was then diluted to 1:750 five sections was counted and multiplied by the number and total ATP content was measured using an ATP corresponding to each section (0-4, labeled from bottom determination kit (A22066, Molecular Probes). Total ATP to top). The flight index was calculated by summing these content was determined by comparing the luminescence values and dividing this sum by the maximum possible measurements for each sample to the ATP standard curve score (four times the number of flies used in the assay). At and normalized to the total number of flies used in the least 100 flies of each genotype were used for a given assay. experiment. Blue native PAGE (BN-PAGE) analysis and in-gel activity Mitochondrial respiratory chain activity assay assay Mitochondrial respiratory chain activity assays were BN-PAGE and in-gel activity assays were performed performed according to a published procedure with sev- according to a previously published protocol . Briefly, eral minor modifications . Briefly, 1000 adult flies were 100 μg of mitochondria prepared from 3-week-old adult homogenized in isolation buffer (5 mM Tris (pH 7.4), flies was solubilized in a buffer containing a digitonin/ 250 mM sucrose, and 2 mM EGTA) with 1% (w/v) fatty protein (w/w) ratio of 8 and subjected to centrifugation at acid free bovine serum albumin. The lysate was subjected 20,000 g for 10 min at 4 °C. Coomassie G-250 was added to centrifugation at 600 g for 10 min to remove cellular to the supernatant and the sample was analyzed by native debris. The supernatant was then subjected to further PAGE. Following electrophoresis, the resulting gel was centrifugation at 7000 g for 10 min to pellet mitochondria. subjected to in-gel activity assays as described below. Mitochondria were washed twice in isolation buffer, The combined complex I and IV in-gel activity assay resuspended in the same buffer, flash frozen in liquid was performed by first incubating the gel in a solution nitrogen, and stored at −80 °C. Roughly 100 µg of mito- containing 1 mg/ml of the complex IV substrate cyto- chondria was used for each assay. Complex I activity was chrome c along with 0.5 mg/ml 3,3′-diaminobenzidine determined spectrophotometrically by monitoring and 45 mM phosphate buffer (pH 7.4) for 40 min. After Official journal of the Cell Death Differentiation Association Pareek et al. Cell Death Discovery (2019) 5:51 Page 12 of 14 the appearance of brown reaction products, the gel was the DNeasy Blood & Tissue kit (Qiagen). A total 5 ng of washed with water and incubated in a solution containing genomic DNA was used as a template to perform qPCR 0.1 mg/ml of the complex I substrate NADH along with using iTaq Universal SYBR Green Supermix (Bio-Rad). 2 mM Tris (pH 7.4) and 2.5 mg/ml nitrotetrazolium blue Mitochondrial DNA levels were estimated by using pri- chloride for 20 min. The reaction was quenched with 10% mers (Supplemental Table 1) to amplify the mt:Cyt-b acetic acid upon the appearance of the violet color indi- gene, and normalized to levels of the nuclear gene Act79b. The relative fold change was determined through the cative of complex I activity. −ΔΔCt 30 The complex II in-gel activity assay was performed by 2 method . incubating the gel in a solution consisting of 5 mM Tris (pH 7.4), 20 mM sodium succinate, 2.5 mg/ml nitrote- RNA isolation and quantitative reverse transcription-PCR trazolium blue chloride, and 0.2 mM phenazine metho- (qRT-PCR) sulfate for 40 min. The reaction was quenched with 10% RNA isolation and qRT-PCR was performed according 24,30,51 acetic acid upon the appearance of the violet color indi- to a previously published procedure . Total RNA cative of complex II activity. was isolated from 40–50 heads obtained from 21-day-old The complex V in-gel activity assay was carried out by adult flies using the Direct-zol RNA MiniPrep kit (Zymo incubating the gel in a solution containing 35 mM Tris, Research). The RNA was reverse transcribed to cDNA 270 mM glycine, 14 mM magnesium sulfate, 10 mM ade- using the iScript cDNA Synthesis Kit (Bio-Rad). For RNA nosine triphosphate, and 0.2% lead (II) nitrate for 16 h. quantification, qRT-PCR experiments were performed The reaction was stopped using 50% methanol upon the using iTaq Universal SYBR Green Supermix and a appearance of silver bands indicative of complex V activity. LightCycler 480 (Roche). Each sample was analyzed in triplicate and normalized to Act79b transcript abundance. −ΔΔCt Immunoblotting The relative fold change was determined by the 2 Fly heads were homogenized inRIPA bufferwithprotease method. All primers used for qRT-PCR are listed in inhibitor cocktail (Roche) for 20 min on ice, and after Supplemental Table 1. centrifugation at 21,000 × g for 20 min the supernatant was collected and subjected to western blot analysis. The anti- In organello translation bodies used were as follows: rabbit anti-LONP1 1:500 De novo labeling of mitochondrial translation products 30,51 (NBP1-81734, Novus Biologicals); mouse anti-Actin was performed as previously described . Approxi- 1:50,000 (MAB1501, Chemicon/Bioscience Research mately 750 µg of mitochondria isolated from third instar Reagents); mouse anti-FLAG 1:1000 (F3165, Sigma); rabbit larvae or adult flies was resuspended in 500 µl of trans- anti-citrate synthase 1:1000 (CISY11-A, Alpha Diagnostics), lation buffer (100 mM mannitol, 10 mM sodium succi- rabbit anti-Hsp60 1:500 (D307, Cell Signaling Technology); nate, 80 mM potassium chloride, 5 mM magnesium and rabbit anti-GRP 75 (H155) 1:1000 (sc-13967, Santa chloride, 1 mM potassium phosphate, 25 mM HEPES (pH Cruz) in Phosphate buffered saline with 0.1% Tween-20 . 7.4), 60 μg/ml all amino acids except methionine, 5 mM The secondary antibodies (anti-mouse HRP and anti-rabbit ATP, 0.2 mM GTP, 6 mM creatine phosphate, and 60 μg/ HRP (Sigma)) were used at a dilution of 1:10,000. Western mL creatine kinase) supplemented with 500 μCi/ml blot images were quantified using ImageJ software (NIH) of S-methionine (Perkin–Elmer). Following incubation and normalized to actin. Each experiment was performed in at 30 °C for 1 h, mitochondria were washed four times triplicate. using isolation buffer and resuspended in SDS sample To analyze unfolded mitochondrial proteins, protein buffer. Roughly 300 µg of mitochondria was subjected to was extracted from fly heads as previously described, SDS-PAGE. Following electrophoresis, the gel was dried except that 0.5% rather than 1% Triton X-100 was used in and exposed to a phosphor screen. The phosphor screen accordance with other work on mitochondrial pro- was scanned using a gel imaging scanner (GE Typhoon 63,64 teins . Antibodies for assaying mitochondrial protein FLA 9000). Mitochondrial translation profile was com- folding status were used as follows: mouse anti-NDUFS3 pared to a previously published study done in Drosophila 1:500 (ab14711, Abcam), mouse anti-Complex V Beta larvae . Roughly 75 µg of mitochondria was loaded on gel 1:2000 (A-21351, Invitrogen), mouse anti-PDH E1α and stained with Coomassie to use as a loading control. (clone 8D10E6) 1:1000 (45-660-0, Fisher Scientific), and rabbit anti-aconitase (ACO2) 1:2000 (AP1936C, Abgent). Mitochondrial ribosomal profiling using sucrose density gradient assay Genomic DNA isolation and mitochondrial DNA copy Mitochondrial ribosomal profiling was performed number estimation according to a previously published procedure with minor Mitochondrial and nuclear DNA was isolated from modifications . Freshly isolated mitochondria (2 mg) were 40–50 heads obtained from 21-day-old adult flies using incubated on ice in lysis buffer (260 mM sucrose, 100 mM Official journal of the Cell Death Differentiation Association Pareek et al. Cell Death Discovery (2019) 5:51 Page 13 of 14 NH Cl, 10 mM MgCl , 30 mM Tris-HCl pH 7.5, 40 U/ml 7. Rugarli, E. I. & Langer, T. Mitochondrial quality control: a matter of life and 4 2 death for neurons. EMBO J. 31, 1336–1349 (2012). Protector RNase Inhibitor, and 1% Triton X-100) supple- 8. Quiros,P.M., Langer, T. & Lopez-Otin,C.New roles for mitochondrialproteases mented with EDTA-free complete protease inhibitor in health, ageing and disease. Nat. Rev. Mol. Cell Biol. 16,345–359 (2015). cocktail (Roche) and PhosSTOP phosphatase inhibitor 9. Pinti, M. et al. Emerging role of Lon protease as a master regulator of mito- chondrial functions. Biochim. Biophys. Acta 1857, 1300–1306 (2016). cocktail (Roche). Mitochondrial lysates were cleared by 10. Pinti, M. et al. Mitochondrial Lon protease at the crossroads of oxidative stress, pelleting the debris through centrifugation at 10,000 g for ageing and cancer. Cell.Mol.LifeSci. 72,4807–4824 (2015). 11. Nystrom, T. Role of oxidative carbonylation in protein quality control and 45 min at 4 °C. The supernatant was then loaded onto a senescence. EMBO J. 24,1311–1317 (2005). 7–47% linear sucrose gradient made in a buffer containing 12. Bota, D. A. & Davies, K. J. Lon protease preferentially degrades oxidized 100 mM NH Cl, 10 mM MgCl , 30 mM Tris-HCl pH 7.5, 4 2 mitochondrial aconitase by an ATP-stimulated mechanism. Nat. Cell Biol. 4, and EDTA-free complete protease inhibitor cocktail 674–680 (2002). 13. Wagner, I.,Arlt, H.,van Dyck,L., Langer,T.& Neupert, W. Molecularchaperones (Roche). Samples were then subjected to centrifugation at cooperate with PIM1 protease in the degradation of misfolded proteins in 39,000 rpm for 6 h at 4 °C. Fractions of 500 μl from the mitochondria. EMBO J. 13,5135–5145 (1994). sucrose gradient sedimentation were collected and RNA 14. Savel’ev, A. S. et al. ATP-dependent proteolysis in mitochondria. m-AAA pro- tease and PIM1 protease exert overlapping substrate specificities and coop- was extracted from each fraction using TRIzol LS Reagent eratewiththe mtHsp70system. J. Biol. Chem. 273,20596–20602 (1998). (Invitrogen) and the Direct-zol RNA MiniPrep kit. RNA 15. Major, T., von Janowsky, B., Ruppert, T., Mogk, A. & Voos, W. Proteomic analysis was reverse transcribed and qRT-PCR analyses were of mitochondrial protein turnover: identification of novel substrate proteins of the matrix protease pim1. Mol. Cell Biol. 26,762–776 (2006). performed to quantify RNAs as described above. 16. Bayot, A. et al. Identification of novel oxidized protein substrates and phy- siological partners of the mitochondrial ATP-dependent Lon-like protease Statistics Pim1. J. Biol. Chem. 285,11445–11457 (2010). 17. Li, L. et al. Changes in specific protein degradation rates in Arabidopsis All findings are presented as mean ± SEM. Unless thaliana reveal multiple roles of Lon1 in mitochondrial protein homeostasis. otherwise stated, statistical significance was calculated Plant J.: Cell Mol. Biol. 89,458–471 (2017). using two-tailed Student’s t-test in GraphPad Prism v5. 18. Song, J. Y.,Marszalek,J.&Craig, E. A. Cysteine desulfurase Nfs1and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold. Proc. Natl Acad. Sci. USA 109,10370–10375 (2012). Acknowledgements 19. Ciesielski, S. J.,Schilke,B., Marszalek, J. &Craig,E.A.Protectionofscaffold We thank Dr. Scott Kennedy (Department of Pathology, University of del protein Isu from degradation by the Lon protease Pim1 as a component of Washington) for assistance with whole genome sequencing of the Lon Fe-S cluster biogenesis regulation. Mol. Biol. Cell. 27,1060–1068 (2016). mutant; Dr. Dana Miller for allowing us to use her lab space for experiments 20. Matsushima, Y., Goto, Y. & Kaguni, L. S. Mitochondrial Lon protease regulates involving radioactive isotopes; and Dr. Evan Eichler, Dr. Christine Queitsch, and mitochondrial DNA copy number and transcription by selective degradation Dr. Stanley Fields for providing access to their lab facilities. We thank Dr. Paul of mitochondrial transcription factor A (TFAM). Proc. Natl Acad. Sci. USA 107, M. Macdonald for providing an antibody against Lrpprc protein. This work was 18410–18415 (2010). supported by a grant from the National Institutes of Health to LP 21. Lu, B. et al. Phosphorylation of human TFAM in mitochondria impairs DNA (R21NS094901). binding and promotes degradation by the AAA+Lon protease. Mol. Cell 49, 121–132 (2013). Conflict of interest 22. Kao, T. Y. et al. Mitochondrial Lon regulates apoptosis through the association The authors declare that they have no conflict of interest. with Hsp60-mtHsp70 complex. Cell Death Dis. 6, e1642 (2015). 23. Strauss, K. A. et al. CODAS syndrome is associated with mutations of LONP1, encoding mitochondrial AAA+Lon protease. Am.J.Hum. Genet. 96, 121–135 Publisher's note (2015). Springer Nature remains neutral with regard to jurisdictional claims in 24. Thomas, R. E.,Andrews,L.A., Burman, J.L., Lin, W. Y. &Pallanck, L. J. PINK1- published maps and institutional affiliations. Parkin pathway activity is regulated by degradation of PINK1 in the mito- chondrial matrix. PLoS Genet. 10, e1004279 (2014). The online version of this article (https://doi.org/10.1038/s41420-018-0110-1) 25. Pareek, G., Thomas, R. E. & Pallanck, L. J. Loss of the Drosophila m-AAA contains supplementary material, which is available to authorized users. mitochondrial protease paraplegin results in mitochondrial dysfunction, shortened lifespan, and neuronal and muscular degeneration. Cell death & Dis. 9, 304 (2018). Received: 7 September 2018 Accepted: 11 September 2018 26. Qi, Y.,Liu,H., Daniels, M. P.,Zhang,G.&Xu,H. Loss ofDrosophila i-AAA protease, dYME1L, causes abnormal mitochondria and apoptotic degenera- tion. Cell Death Differ. 23,291–302 (2016). 27. Quiros, P. M. et al. ATP-dependent Lon protease controls tumor bioenergetics References by reprogramming mitochondrial activity. Cell Rep. 8,542–556 (2014). 1. Kauppila,T.E.S., Kauppila,J.H.K.&Larsson, N. G. Mammalianmitochondria 28. Garcia, C. J.,Khajeh, J.,Coulanges,E., Chen,E. I.&Owusu-Ansah, E. Regulation and aging: an update. Cell Metab. 25,57–71 (2017). of mitochondrial complex I biogenesis in Drosophila flight muscles. Cell Rep. 2. Sun, N., Youle,R.J.& Finkel, T.The mitochondrialbasis of aging. Mol. Cell 61, 20,264–278 (2017). 654–666 (2016). 29. Vincow, E. S. et al. The PINK1-Parkin pathway promotes both mitophagy and 3. Pickrell, A. M. & Youle, R. J. The roles of PINK1, parkin, and mitochondrial fidelity selective respiratory chain turnover in vivo. Proc. Natl Acad. Sci. USA 110, in Parkinson’sdisease. Neuron 85,257–273 (2015). 6400–6405 (2013). 4. Gustafsson,C.M., Falkenberg, M.&Larsson, N. G. Maintenance and 30. Baggio, F. et al. Drosophila melanogaster LRPPRC2 is involved in coordination expression of mammalian mitochondrial DNA. Annu. Rev. Biochem. 85, of mitochondrial translation. Nucleic Acids Res. 42,13920–13938 (2014). 133–160 (2016). 31. Szczepanowska, K. et al. CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels. EMBO J. 35,2566–2583 (2016). 5. Hallberg, B. M. &Larsson,N.G.Making proteins in the powerhouse. Cell Metab. 32. Camara, Y. et al. MTERF4 regulates translation by targeting the methyl- 20, 226–240 (2014). transferase NSUN4 to the mammalian mitochondrial ribosome. Cell Metab. 13, 6. Patron,M., Sprenger,H.G.&Langer,T.m-AAA proteases, mitochondrialcal- 527–539 (2011). cium homeostasis and neurodegeneration. Cell Res. 28, 296–306 (2018). Official journal of the Cell Death Differentiation Association Pareek et al. Cell Death Discovery (2019) 5:51 Page 14 of 14 33. Dogan,S.A.et al. Tissue-specific loss of DARS2 activates stress responses 49. Siegelin, M. D. et al. Exploiting the mitochondrial unfolded protein response independently of respiratory chain deficiency in the heart. Cell Metab. 19, for cancer therapy in mice and human cells. J. Clin. Invest. 121,1349–1360 458–469 (2014). (2011). 34. Bernstein, S. H. et al. The mitochondrial ATP-dependent Lon protease: a novel 50. Munch,C.& Harper,J.W.Mitochondrial unfolded proteinresponsecontrols target in lymphoma death mediated by the synthetic triterpenoid CDDO and matrix pre-RNA processing and translation. Nature 534,710–713 (2016). its derivatives. Blood 119,3321–3329 (2012). 51. Bratic, A. et al. The bicoid stability factor controls polyadenylation and 35. Suzuki, C. K., Suda, K., Wang, N. & Schatz, G. Requirement for the yeast gene expression of specific mitochondrial mRNAs in Drosophila melanogaster. PLoS LON in intramitochondrial proteolysis and maintenance of respiration. Science Genet. 7, e1002324 (2011). 264, 891 (1994). 52. Rackham, O. et al. Hierarchical RNA Processing Is Required for Mitochondrial 36. Bezawork-Geleta, A., Brodie,E.J., Dougan,D.A.&Truscott, K.N. LON is the Ribosome Assembly. Cell Rep. 16,1874–1890 (2016). master protease that protects against protein aggregation in human mito- 53. Bezawork-Geleta,A., Saiyed,T., Dougan, D. A.& Truscott, K.N. Mitochondrial chondria through direct degradation of misfolded proteins. Sci. Rep. 5, 17397 matrix proteostasis is linked to hereditary paraganglioma: LON-mediated (2015). turnover of the human flavinylation factor SDH5 is regulated by its interaction 37. Ron,D.& Walter,P.Signalintegration in the endoplasmic reticulum unfolded with SDHA. FASEB J. 28,1794–1804 (2014). protein response. Nat. Rev. Mol. Cell Biol. 8,519–529 (2007). 54. Matsushima, Y. et al. Drosophila protease ClpXP specifically degrades 38. Walter, P. & Ron, D. The unfolded protein response: from stress pathway to DmLRPPRC1 controlling mitochondrial mRNA and translation. Sci. Rep. 7,8315 homeostatic regulation. Science 334, 1081–1086 (2011). (2017). 39. Shpilka, T. & Haynes, C. M. The mitochondrial UPR: mechanisms, physiological 55. Siira, S. J. et al. LRPPRC-mediated folding of the mitochondrial transcriptome. functions and implications in ageing. Nat. Rev. Mol. Cell Biol. 19,109–120 Nat. Commun. 8,1532 (2017). (2018). 56. Gilkerson, R. et al. The mitochondrial nucleoid: integrating mitochondrial DNA 40. Pakos-Zebrucka, K. et al. The integrated stress response. EMBO Rep. 17, into cellular homeostasis. Cold Spring Harb. Perspect. Biol. 5, a011080 (2013). 1374–1395 (2016). 57. Gratz,S.J.etal. Highly specificand efficient CRISPR/Cas9-catalyzed homology- 41. Balch, W. E., Morimoto, R. I., Dillin, A. & Kelly, J. W. Adapting proteostasis for directed repair in Drosophila. Genetics 196,961–971 (2014). disease intervention. Science 319, 916–919 (2008). 58. Gargano,J.W., Martin,I., Bhandari,P.&Grotewiel, M. S. Rapiditerative negative 42. Lamech, L. T. & Haynes, C. M. The unpredictability of prolonged activation of geotaxis (RING): a new method for assessing age-related locomotor decline in stress response pathways. J. Cell Biol. 209, 781–787 (2015). Drosophila. Exp. Gerontol. 40,386–395 (2005). 43. Akerfelt, M., Morimoto, R. I. & Sistonen, L. Heat shock factors: integrators of 59. Greene, J. C. et al. Mitochondrial pathology and apoptotic muscle degen- cell stress, development and lifespan. Nat. Rev. Mol. Cell Biol. 11,545–555 eration in Drosophila parkin mutants. Proc. Natl Acad. Sci. USA 100,4078–4083 (2010). (2003). 44. Protter, D. S. & Parker, R. Principles and properties of stress granules. Trends Cell 60. Burman, J. L. et al. A Drosophila model of mitochondrial disease caused by a Biol. 26,668–679 (2016). complex I mutation that uncouples proton pumping from electron transfer. 45. Nargund, A. M., Fiorese, C. J., Pellegrino, M. W., Deng, P. & Haynes, C. M. Dis. Models Mech. 7, 1165–1174 (2014). Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 61. Tennessen,J.M., Barry,W.E., Cox, J. &Thummel, C. S. Methodsfor studying promotes OXPHOS recovery during the UPR(mt). Mol. Cell 58, 123–133 (2015). metabolism in Drosophila. Methods 68,105–115 (2014). 46. Nargund,A.M., Pellegrino, M. W.,Fiorese, C.J., Baker, B. M. &Haynes, C. M. 62. Jha,P., Wang,X.& Auwerx,J.Analysis ofmitochondrial respiratorychain Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR acti- supercomplexes using blue native polyacrylamide gel electrophoresis (BN- vation. Science 337,587–590 (2012). PAGE). Curr. Protoc. Mouse Biol. 6,1–14 (2016). 47. Zhao, Q. et al. A mitochondrial specific stress response in mammalian cells. 63. Pimenta de Castro, I. et al. Genetic analysis of mitochondrial protein misfolding EMBO J. 21, 4411–4419 (2002). in Drosophila melanogaster. Cell Death Differ. 19,1308–1316 (2012). 48. Owusu-Ansah, E., Song, W. & Perrimon, N. Muscle mitohormesis 64. Davis,M.Y.etal. Glucocerebrosidase deficiency in Drosophila results in alpha- promotes longevity via systemic repression of insulin signaling. Cell 155, synuclein-independent protein aggregation and neurodegeneration. PLoS 699–712 (2013). Genet. 12, e1005944 (2016). Official journal of the Cell Death Differentiation Association

Journal

Cell Death Discovery – Springer Journals

Published: Oct 22, 2018

There are no references for this article.