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• W2024952866 abstract "Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting the motor neurons. The majority of familial forms of ALS are caused by mutations in the Cu,Zn-superoxide dismutase (SOD1). In mutant SOD1 spinal cord motor neurons, mitochondria develop abnormal morphology, bioenergetic defects, and degeneration. However, the mechanisms of mitochondrial toxicity are still unclear. One possibility is that mutant SOD1 establishes aberrant interactions with nuclear-encoded mitochondrial proteins, which can interfere with their normal trafficking from the cytosol to mitochondria. Lysyl-tRNA synthetase (KARS), an enzyme required for protein translation that was shown to interact with mutant SOD1 in yeast, is a good candidate as a target for interaction with mutant SOD1 at the mitochondrion in mammals because of its dual cytosolic and mitochondrial localization. Here, we show that in mammalian cells mutant SOD1 interacts preferentially with the mitochondrial form of KARS (mitoKARS). KARS-SOD1 interactions occur also in the mitochondria of the nervous system in transgenic mice. In the presence of mutant SOD1, mitoKARS displays a high propensity to misfold and aggregate prior to its import into mitochondria, becoming a target for proteasome degradation. Impaired mitoKARS import correlates with decreased mitochondrial protein synthesis. Ultimately, the abnormal interactions between mutant SOD1 and mitoKARS result in mitochondrial morphological abnormalities and cell toxicity. mitoKARS is the first described member of a group of mitochondrial proteins whose interaction with mutant SOD1 contributes to mitochondrial dysfunction in ALS. Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting the motor neurons. The majority of familial forms of ALS are caused by mutations in the Cu,Zn-superoxide dismutase (SOD1). In mutant SOD1 spinal cord motor neurons, mitochondria develop abnormal morphology, bioenergetic defects, and degeneration. However, the mechanisms of mitochondrial toxicity are still unclear. One possibility is that mutant SOD1 establishes aberrant interactions with nuclear-encoded mitochondrial proteins, which can interfere with their normal trafficking from the cytosol to mitochondria. Lysyl-tRNA synthetase (KARS), an enzyme required for protein translation that was shown to interact with mutant SOD1 in yeast, is a good candidate as a target for interaction with mutant SOD1 at the mitochondrion in mammals because of its dual cytosolic and mitochondrial localization. Here, we show that in mammalian cells mutant SOD1 interacts preferentially with the mitochondrial form of KARS (mitoKARS). KARS-SOD1 interactions occur also in the mitochondria of the nervous system in transgenic mice. In the presence of mutant SOD1, mitoKARS displays a high propensity to misfold and aggregate prior to its import into mitochondria, becoming a target for proteasome degradation. Impaired mitoKARS import correlates with decreased mitochondrial protein synthesis. Ultimately, the abnormal interactions between mutant SOD1 and mitoKARS result in mitochondrial morphological abnormalities and cell toxicity. mitoKARS is the first described member of a group of mitochondrial proteins whose interaction with mutant SOD1 contributes to mitochondrial dysfunction in ALS. Amyotrophic lateral sclerosis (ALS) 2The abbreviations used are: ALS, amyotrophic lateral sclerosis; SOD1, Cu,Zn-superoxide dismutase; hSOD1, human SOD1; KARS or LysRS, lysyl-tRNA synthetase; mitoKARS, mitochondrial lysyl-tRNA synthetase; cytoKARS, cytosolic lysyl-tRNA synthetase; pre-MSK1p, yeast mitoKARS precursor; mitoGFP, mitochondrially targeted green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tim23, translocase of inner membrane 23-kDa subunit; Hsp70, heat shock protein 70; MnSOD, manganese-superoxide dismutase; PBS, phosphate-buffered saline; WT, wild type; IP, immunoprecipitation; co-IP, co-immunoprecipitation; UPS, ubiquitin-proteasome system; mtDNA, mitochondrial DNA. is a progressive neurodegenerative disorder of motor neurons that results in paralysis and death within five years of diagnosis. Approximately 10% of ALS cases are inherited, of which 20% are associated with mutations in the Cu,Zn-superoxide dismutase, SOD1. SOD1 is a free radical scavenging enzyme, but because many SOD1 mutations do not affect the enzymatic activity and the disease has an autosomal dominant transmission, a toxic gain of function of the mutant protein has been postulated. SOD1 is abundantly expressed in the cytosol, but a proportion of mutant SOD1 is also associated with mitochondria, where its aggregation could have pathological consequences (1Higgins C.M. Jung C. Ding H. Xu Z. J. Neurosci. 2002; 22: RC215Crossref PubMed Google Scholar, 2Jaarsma D. Rognoni F. van Duijn W. Verspaget H.W. Haasdijk E.D. Holstege J.C. Acta Neuropathol. 2001; 102: 293-305Crossref PubMed Scopus (224) Google Scholar, 3Liu J. Lillo C. Jonsson P.A. Velde C.V. Ward C.M. Miller T.M. Subramaniam J.R. Rothstein J.D. Marklund S. Andersen P.M. Brannstrom T. Gredal O. Wong P.C. Williams D.S. Cleveland D.W. Neuron. 2004; 43: 5-17Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 4Mattiazzi M. D'Aurelio M. Gajewski C.D. Martushova K. Kiaei M. Beal M.F. Manfredi G. J. Biol. Chem. 2002; 277: 29626-29633Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 5Okado-Matsumoto A. Fridovich I. J. Biol. Chem. 2001; 276: 38388-38393Abstract Full Text Full Text PDF PubMed Scopus (813) Google Scholar, 6Vijayvergiya C. Beal M.F. Buck J. Manfredi G. J. Neurosci. 2005; 25: 2463-2470Crossref PubMed Scopus (196) Google Scholar, 7Deng H.X. Shi Y. Furukawa Y. Zhai H. Fu R. Liu E. Gorrie G.H. Khan M.S. Hung W.Y. Bigio E.H. Lukas T. Dal Canto M.C. O'Halloran T.V. Siddique T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7142-7147Crossref PubMed Scopus (352) Google Scholar). Transgenic mice expressing mutant human SOD1 (hSOD1) develop mitochondrial degeneration in motor neurons (8Dal Canto M.C. Gurney M.E. Brain Res. 1995; 676: 25-40Crossref PubMed Scopus (384) Google Scholar, 9Wong P.C. Pardo C.A. Borchelt D.R. Lee M.K. Copeland N.G. Jenkins N.A. Sisodia S.S. Cleveland D.W. Price D.L. Neuron. 1995; 14: 1105-1116Abstract Full Text PDF PubMed Scopus (1258) Google Scholar), whose appearance coincides with the onset of symptoms (10Kong J. Xu Z. J. Neurosci. 1998; 18: 3241-3250Crossref PubMed Google Scholar). Furthermore, mutant hSOD1 transgenic mice develop dysfunction of mitochondrial respiration and ATP synthesis (4Mattiazzi M. D'Aurelio M. Gajewski C.D. Martushova K. Kiaei M. Beal M.F. Manfredi G. J. Biol. Chem. 2002; 277: 29626-29633Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 11Jung C. Higgins C.M. Xu Z. J. Neurosci. Methods. 2002; 114: 165-172Crossref PubMed Scopus (61) Google Scholar, 12Kirkinezos I.G. Bacman S.R. Hernandez D. Oca-Cossio J. Arias L.J. Perez-Pinzon M.A. Bradley W.G. Moraes C.T. J. Neurosci. 2005; 25: 164-172Crossref PubMed Scopus (162) Google Scholar). In addition, we have demonstrated that this bioenergetic failure results in impaired mitochondrial calcium uptake in the spinal cord and brain of mutant hSOD1 mice (13Damiano M. Starkov A.A. Petri S. Kipiani K. Kiaei M. Mattiazzi M. Flint Beal M. Manfredi G. J. Neurochem. 2006; 96: 1349-1361Crossref PubMed Scopus (193) Google Scholar). Despite the evidence that mutant SOD1 causes mitochondrial dysfunction (14Hervias I. Beal M.F. Manfredi G. Muscle Nerve. 2006; 33: 598-608Crossref PubMed Scopus (98) Google Scholar), the molecular mechanisms underlying the mitochondrial damage remain to be identified. The large majority of mitochondrial protein components are nuclear-encoded, synthesized in the cytosol, and imported into mitochondria through specialized import machineries. Thus, one hypothesis for mutant SOD1 toxicity involves aberrant interactions of mutant SOD1 with mitochondrial proteins (7Deng H.X. Shi Y. Furukawa Y. Zhai H. Fu R. Liu E. Gorrie G.H. Khan M.S. Hung W.Y. Bigio E.H. Lukas T. Dal Canto M.C. O'Halloran T.V. Siddique T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7142-7147Crossref PubMed Scopus (352) Google Scholar), resulting in disruption of their normal folding and import (3Liu J. Lillo C. Jonsson P.A. Velde C.V. Ward C.M. Miller T.M. Subramaniam J.R. Rothstein J.D. Marklund S. Andersen P.M. Brannstrom T. Gredal O. Wong P.C. Williams D.S. Cleveland D.W. Neuron. 2004; 43: 5-17Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Interactions involving mutant SOD1 have been reported with proteins that may affect directly or indirectly mitochondria, including heat shock proteins and Bcl-2 (15Okado-Matsumoto A. Fridovich I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9010-9014Crossref PubMed Scopus (214) Google Scholar, 16Pasinelli P. Belford M.E. Lennon N. Bacskai B.J. Hyman B.T. Trotti D. Brown Jr., R.H. Neuron. 2004; 43: 19-30Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). In a yeast two-hybrid screen, lysyl-tRNA synthetase (KARS), an enzyme required for protein synthesis, was found to interact with mutant but not with wild type (WT) SOD1 (17Kunst C.B. Mezey E. Brownstein M.J. Patterson D. Nat. Genet. 1997; 15: 91-94Crossref PubMed Scopus (109) Google Scholar). KARS is a potential candidate for abnormal interactions with SOD1 affecting mitochondria, because it exists both as a cytosolic (cytoKARS) and as a mitochondrially imported (mitoKARS) enzyme. Here, we investigate the interactions between mutant hSOD1 and KARS in mammalian cells and their consequences on mitochondrial integrity and cell viability. We find that as a consequence of aberrant interactions with mutant hSOD1, mitoKARS misfolds prior to or during its import into mitochondria and becomes targeted for proteasomal degradation. Mutant hSOD1-mitoKARS interactions result in the formation of high molecular weight protein aggregates that correlate with impaired mtDNA-encoded protein synthesis, mitochondrial morphological abnormalities, and decreased cell survival. Expression Plasmids—Human mitoKARS cDNA was cloned in pEF/Myc/cyto (Invitrogen) as described previously (18Tolkunova E. Park H. Xia J. King M.P. Davidson E. J. Biol. Chem. 2000; 275: 35063-35069Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Human cytoKARS with a C-terminal FLAG epitope tag and hSOD1 cDNAs (wild type and G93A and G85R mutants) were cloned into pCIneo (Promega, Madison, WI) and pcDNA3.0 (Invitrogen), respectively. Mitochondrial GFP (mitoGFP) was a gift of Dr. Rosario Rizzuto (19Rizzuto R. Brini M. Pizzo P. Murgia M. Pozzan T. Curr. Biol. 1995; 5: 635-642Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar) (University of Ferrara, Ferrara, Italy). Cell Culture, Transfection, and Proteasome Inhibition—COS-7 cells (ATCC, Manassas, VA) were cultured in advanced Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 2% fetal bovine serum (Cellgro, Herndon, VA) and Gluta-max (Invitrogen), in 5% CO2 at 37 °C. Transfections with plasmids encoding KARS and hSOD1, either individually or in combination, were performed using FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's instructions. For proteasome inhibition experiments, the cells were treated for 16 h with 20 μm (complete inhibition) or 75 nm (partial inhibition) of MG132 (Sigma). hSOD1 Transgenic Mice—Transgenic mice expressing WT (N1029) or G93A (20Gurney M.E. Pu H. Chiu A.Y. Dal Canto M.C. Polchow C.Y. Alexander D.D. Caliendo J. Hentati A. Kwon Y.W. Deng H.X. et al.Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3494) Google Scholar) hSOD1 were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred at the Weill Medical College of Cornell University animal facility. All of the animal procedures were approved by the Animal Care and Use Committee of the Weill Medical College of Cornell University. Cell Imaging by Immunocytochemistry—Forty-eight hours post-transfection, the cells grown on glass coverslips were washed in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde. After three washes in PBS, the cells were permeabilized with 0.1% Triton X-100, and blocked in PBS containing 1% bovine serum albumin and 10% normal goat serum. The cells were incubated with primary antibodies diluted in blocking buffer for 2 h with gentle agitation. Fluorescently labeled secondary antibodies were diluted in blocking buffer and applied to cells for 1 h. The cells were washed in PBS three times after primary and secondary antibody incubations. All of the steps were performed at room temperature. The following antibodies were used: monoclonal hSOD1 (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal SOD1 (Stressgen, Victoria, Canada), FLAG-M2 (Sigma), monoclonal Myc (Upstate, Lake Placid, NY or Abcam, Cambridge, MA), polyclonal Myc (Sigma), Hsp70 (Stressgen), and MnSOD (Stressgen). Immunostained cells were imaged with a Zeiss LSM 510 laser scanning confocal microscope with a 63× Plan Apochromat oil immersion lens with aperture 1.4 using a photomultiplier (Carl Zeiss MicroImaging, Inc.). A series of z sections were taken spanning the thickness of the cell with intervals between sections set at 0.5 μm. z stack images were projected onto a single plane using the LSM Image Browser software (Carl Zeiss MicroImaging, Inc.), and digital magnification was 2× (total magnification was 126×). Cell Viability Assay—COS cells grown in 48-well plates were transfected with hSOD1 (WT, G93A, G85R, or empty vector) with or without mitoKARS. Twenty-four hours later, the cells were washed once in PBS and incubated with 2.5 μm calcein AM (Invitrogen) for 10 min at room temperature. Fluorescence was measured at 485-nm excitation and 535-nm emission in an HTS 7000 plus plate reader (Packard Instrument Company, Downers Grove, IL) with background subtraction. Cell and Tissue Fractionation—COS cells transfected with hSOD1 and KARS were fractionated into cytosolic and enriched mitochondrial fractions according to established protocols (21Pallotti F. Lenaz G. Methods Cell Biol. 2001; 65: 1-35Crossref PubMed Google Scholar). Mouse brain and spinal cord mitochondria were isolated and purified in a Ficoll gradient as described previously (6Vijayvergiya C. Beal M.F. Buck J. Manfredi G. J. Neurosci. 2005; 25: 2463-2470Crossref PubMed Scopus (196) Google Scholar) with minor modifications. The tissue homogenization buffer contained 20 mm Hepes instead of Tris-HCl to allow for chemical cross-linking. Immunoprecipitation and Western Blot Analyses—Cells or tissue fractions were cross-linked with 2 mm dithiobis(succinimidyl propionate) (Pierce) dissolved in Me2SO for 30 min at room temperature, followed by the addition of 20 mm Tris (pH 7.6) and incubation for 15 min to stop the reaction. For cells, the samples were washed three times in PBS, lysed in RIPA buffer containing 20 mm Tris, pH 7.4, 150 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and a protease inhibitor mixture (Roche), and cleared by centrifugation at 10,000 × g for 5 min at 4 °C. The supernatants were incubated overnight at 4 °C with protein G-Sepharose beads (Zymed Laboratories Inc., S. San Francisco, CA), which had been preadsorbed with appropriate antibodies for 2 h at room temperature and collected by brief centrifugation. The following day, the beads were washed three times in RIPA buffer and boiled for 10 min in Laemmli buffer containing 50 mm dithiothreitol prior to electrophoresis. For mouse tissue, the samples were solubilized with 1% Triton X-100 for 15 min at room temperature. Immunoprecipitation was performed as above, except that the buffer contained 10 mm Tris, pH 7.4, 320 mm sucrose, 150 mm NaCl, 5 mm MgCl2, and 1% Triton X-100. Immunoprecipitated samples were separated by standard SDS-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked in 5% milk (in Tris-buffered saline with 0.1% Tween 20) for 1 h, followed by primary antibody incubation overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies were applied for 1 h at room temperature, and immunoreactive bands were revealed with the enhanced chemiluminescence reagent (Pierce). The following antibodies were used for immunoprecipitation and detection of proteins: LysRS against human KARS (22Cen S. Javanbakht H. Kim S. Shiba K. Craven R. Rein A. Ewalt K. Schimmel P. Musier-Forsyth K. Kleiman L. J. Virol. 2002; 76: 13111-13115Crossref PubMed Scopus (69) Google Scholar), polyclonal SOD1 (Calbiochem, La Jolla, CA), ubiquitin (Chemicon Millipore, Billerica, MA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Abcam), Tim23 (BD Biosciences, La Jolla, CA), Myc, and FLAG-M2. Blue Native Gel Electrophoresis—COS cells were transfected with mitoKARS with or without hSOD1 (WT, G93A, and G85R). The mitochondria were solubilized, and native protein complexes were separated by blue native gel electrophoresis and transferred to polyvinylidene difluoride membranes, as described previously (23D'Aurelio M. Gajewski C.D. Lenaz G. Manfredi G. Hum. Mol. Genet. 2006; 15: 2157-2169Crossref PubMed Scopus (105) Google Scholar). The membranes were blocked in 5% milk in Tris-buffered saline-Tween for 8 h and immunoprobed with LysRS or complex III core II subunit (Invitrogen) antibodies. Filter Trap Assays—The filter trap assay was described previously (6Vijayvergiya C. Beal M.F. Buck J. Manfredi G. J. Neurosci. 2005; 25: 2463-2470Crossref PubMed Scopus (196) Google Scholar). Briefly, cytosolic and mitochondrial fractions from COS cells or tissues were incubated with 0.5% Triton X-100 for 15 min on ice. The samples were vacuum-filtered through 0.22-μm cellulose acetate membranes (GE Osmonics, Trevose, PA) using a 96-well dot blot apparatus (Bio-Rad). The membranes were washed and immunodetected with appropriate primary and secondary antibodies as described above. In a set of experiments, proteinase K treatment was performed on mitochondrial fractions from COS cells co-transfected with mutant hSOD1 and mitoKARS prior to performing the filter trap assay. Twenty μg of the samples were treated with 20 μg/ml proteinase K on ice for 30 min. Proteinase K was inactivated with 2 mm phenylmethanesulfonyl fluoride for 15 min. Mitochondrial Protein Synthesis Analysis—The rate of mitochondrial protein synthesis was measured by pulse-labeling experiments with [35S]methionine according to the method of Chomyn (24Chomyn A. Methods Enzymol. 1996; 264: 197-211Crossref PubMed Google Scholar). COS cells were co-transfected with WT or G85R hSOD1 and mitoKARS. 48 h post-transfection, the cells were labeled with [35S]methionine (0.2 mCi of 1,175 Ci/mmol/plate) for 30 min in the presence of the cytoplasmic translation inhibitor emetine (50 μg/ml). The labeled cells were trypsinized, washed, and treated with 1% SDS. Samples containing 50 μg of protein were electrophoresed through a 15–20% exponential SDS-polyacrylamide gradient gel. The gel was dried and exposed to a phosphorimaging screen, and selected radioactive bands corresponding to mtDNA-encoded peptides ND5, ND1, and A6 were analyzed with a Cyclone phosphorimaging device (Packard Instrument Company). Cell lysates were also subjected to Western blot to determine the expression levels of mitoKARS and hSOD1 using antibodies against Myc and SOD1, as well as loading controls for nuclear-encoded mitochondrial protein, Tim23, and the cytosolic protein, GAPDH. KARS Expression and Subcellular Localization in COS Cells—Expression studies with the two isoforms of KARS were conducted using plasmid vectors coding for human mitoKARS or cytoKARS tagged at the C-termini with Myc and FLAG epitopes, respectively (Fig. 1A). Upon transient transfection in COS cells, mitoKARS is distributed to the mitochondria, as shown by co-localization of Myc immunostaining with the mitochondrial matrix protein MnSOD (Fig. 1B). In contrast, the cytoKARS protein lacking a mitochondrial targeting signal is expressed diffusely in the cell and co-localizes with the cytoplasmic chaperone protein, Hsp70 (Fig. 1C). Subcellular localization of KARS was further analyzed by immunoblot of cytosolic and mitochondrial fractions. mitoKARS is detected as the 69-kDa unprocessed form (i.e. still containing the N-terminal mitochondrial targeting signal) in the cytosolic fraction and predominantly as the 64-kDa processed form in the mitochondrial fraction (Fig. 1D). As expected, cytoKARS is localized only in the cytosolic fraction (Fig. 1D). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Tim23 were used as the cytosolic and mitochondrial markers, respectively. cytoKARS is thought to form a dimer, which is part of a large protein complex containing multiple aminoacylating enzymes (25Mirande M. Le Corre D. Waller J.P. Eur. J. Biochem. 1985; 147: 281-289Crossref PubMed Scopus (81) Google Scholar, 26Robinson J.C. Kerjan P. Mirande M. J. Mol. Biol. 2000; 304: 983-994Crossref PubMed Scopus (102) Google Scholar), but the functional structure of mitoKARS has not yet been defined. Thus, to assess whether also mitoKARS exists in multimeric complexes, we performed a blue native gel analysis, which detected a single band of ∼150 kDa, presumably corresponding to a mitoKARS dimer (Fig. 1E). mitoKARS Co-immunoprecipitates with SOD1—Co-immunoprecipitation (co-IP) experiments were carried out to investigate the interaction between hSOD1 and mitoKARS. Because KARS was shown to interact with mutant SOD1 in yeast (17Kunst C.B. Mezey E. Brownstein M.J. Patterson D. Nat. Genet. 1997; 15: 91-94Crossref PubMed Scopus (109) Google Scholar), we tested two different mutants of hSOD1, G93A and G85R, and compared them with WT hSOD1. The two mutants differ by their structural and biochemical properties, because the G93A hSOD1 is folded into a stable and active protein, whereas the G85R hSOD1 is highly unstable and lacks detectable dismutase activity. mitoKARs is pulled down by the SOD1 antibody only in cells expressing mutant hSOD1. Reverse co-IP with the Myc antibody pulls down G93A and G85R mutant hSOD1 efficiently, whereas only a small amount of WT hSOD1 is detected (Fig. 2A, top left panels), despite the fact that similar amounts of the three forms of hSOD1 are expressed in cell lysates (Fig. 2A, bottom left panels). As expected, a negative control co-IP in the absence of antibodies failed to pull down KARS or SOD1 (Fig. 2A, top right panels). Under the same experimental conditions, expression of mutant or WT hSOD1 together with cytoKARS did not result in detectable co-IP of the two proteins (data not shown), suggesting that SOD1 interacts selectively with mitoKARS and not with cytoKARS. To assess whether SOD1-KARS interactions take place in vivo in a disease-relevant tissue with endogenous expression of KARS, we investigated mitochondrial and cytosolic fractions from brain and spinal cord of WT and G93A hSOD1 transgenic mice. When SOD1 was pulled down, co-immunoprecipitated KARS was detected using the LysRS antibody, which recognizes both forms of KARS. This antibody was raised against human KARS (22Cen S. Javanbakht H. Kim S. Shiba K. Craven R. Rein A. Ewalt K. Schimmel P. Musier-Forsyth K. Kleiman L. J. Virol. 2002; 76: 13111-13115Crossref PubMed Scopus (69) Google Scholar), but it reacts also with mouse KARS. Both WT and G93A SOD1 pulled down endogenous KARS more efficiently in the mitochondrial fractions as compared with the cytosolic ones, both in brain and spinal cord (Fig. 2B). Relative to the amount of immunoprecipitated SOD1, KARS is pulled down more efficiently in G93A than in WT spinal cord mitochondria, suggesting that KARS may interact more strongly with mutant than WT hSOD1. SOD1 Induces the Formation of Aggregates Containing mitoKARS—Mutant SOD1 forms aggregates in the cytosol and mitochondria (6Vijayvergiya C. Beal M.F. Buck J. Manfredi G. J. Neurosci. 2005; 25: 2463-2470Crossref PubMed Scopus (196) Google Scholar, 27Wang J. Xu G. Borchelt D.R. Neurobiol. Dis. 2002; 9: 139-148Crossref PubMed Scopus (176) Google Scholar). Therefore, we hypothesized that aberrant protein-protein interaction with mutant SOD1 would lead to recruitment of KARS into aggregates. COS cells were co-transfected with WT or mutant hSOD1 and either cyto or mitoKARS and cytosolic and mitochondrial fractions were subjected to size exclusion filter trap assays. Using the LysRS antibody, we find that cells expressing mitoKARS contain unfilterable aggregates in the mitochondrial fraction. On the other hand, in both the cytosolic and mitochondrial fractions of cells transfected with cytoKARS, unfilterable aggregates are virtually undetectable (Fig. 2C). This is not due to lower expression of cytoKARS than mitoKARS, as shown by Western blot of the lysates (Fig. 2D). We note that the expression of mitoKARS alone results in aggregation (Fig. 2C), but co-expression with hSOD1 clearly increases mitoKARS aggregation. In the same mitoKARS and SOD1 transfected cell samples, we also confirm the presence of mitoKARS aggregates associated with mitochondria using the Myc antibody and show that mutant hSOD1 increases KARS aggregation more than WT hSOD1 (Fig. 2E). Filter trap assays of cytosolic and mitochondrial fractions from brain and spinal cord of transgenic mice show unfilterable protein aggregates containing KARS, which are more abundant in the mitochondrial fraction of G93A mice (Fig. 2F, top panel). These aggregates correlate with the presence of unfilterable SOD1 aggregates (Fig. 2F, bottom panel), indicating that also in vivo KARS aggregates are preferentially associated with mitochondria containing mutant hSOD1. mitoKARs Aggregates Are Associated with the Outer Surface of Mitochondria—The presence of mitoKARS aggregates in the cytoplasmic fractions (Fig. 2, C and E) suggests that a portion of the protein aggregates prior to being imported. To determine the site of formation of the mitoKARS aggregates, we treated intact mitochondria with proteinase K prior to the filter trap assay. The majority of mitoKARS aggregates are digested by proteinase K, whereas MnSOD, a protein of the mitochondrial matrix, is protected from digestion (Fig. 3A), suggesting that aggregation of mitoKARS occurs mostly on the external surface of the mitochondrial outer membrane. To test whether mutant hSOD1 interferes with mitoKARS import, we correlated the amount of mitoKARS associated with mitochondria with that of total cellular mitoKARS. Mitochondrial mitoKARS was normalized by the content of Tim23, a marker of the inner membrane. Total cellular mitoKARS was normalized by the content of the cytosolic protein GAPDH. We find less mitoKARS associated with mitochondria in mutant hSOD1 cells, as compared with cells expressing WT hSOD1 (Fig. 3, B and C). To determine whether reduced mitoKARS import affects normal mitochondrial protein synthesis, we performed a [35S]methionine pulse labeling of mitochondrial DNA (mtDNA) synthesized proteins in cells expressing KARS and WT or G85R hSOD1. We find a generalized decrease in mitochondrial protein synthesis in cells expressing G85R hSOD1 (Fig. 3D). Quantification by phosphorimaging of selected labeled mitochondrial peptides revealed approximately a 25% reduction in the translation of ND1, ND5, and A6 (data not shown). The same samples were also analyzed by Western blot to detect GAPDH and Tim23 as protein loading controls and hSOD1 and mitoKARS to confirm transgene expression (Fig. 3E). These results suggest that mutant hSOD1 interaction affects mitochondrial function through an impairment of protein synthesis. Blue native gel electrophoresis to detect assembled mitochondrial respiratory chain complexes detected a defect in complex III, which paralleled a reduction in the content of dimeric mitoKARS in cells expressing G85R mutant hSOD1 (Fig. 3F). The reduction in complex III is likely the result of decreased synthesis of cytochrome b, which is mtDNA-encoded. We looked at the nuclear encoded mitochondrial proteins VDAC, Tim 23, and Hsp60 by Western blot in the samples used for blue native gels to control for protein loading (Fig. 3G). Nonimported mitoKARS Is Degraded through the Ubiquitin-Proteasome System—The ubiquitin-proteasome system (UPS) has been linked to ALS pathogenesis (reviewed in Ref. 28Kabashi E. Durham H.D. Biochim. Biophys. Acta. 2006; 1762: 1038-1050Crossref PubMed Scopus (78) Google Scholar). Among many other functions, UPS is involved in regulating mitochondrial protein import (29Pearce D.A. Sherman F. J. Biol. Chem. 1997; 272: 31829-31836Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and mutations in components of the UPS can affect mitochondrial morphology in yeast (30Fisk H.A. Yaffe M.P. J. Cell Biol. 1999; 145: 1199-1208Crossref PubMed Scopus (161) Google Scholar, 31Rinaldi T. Pick E. Gambadoro A. Zilli S. Maytal-Kivity V. Frontali L. Glickman M.H. Biochem. J. 2004; 381: 275-285Crossref PubMed Scopus (50) Google Scholar). In addition, proteins involved in tRNA import, including the precursor of mitoKARS (pre-MSK1p), interact with components of the UPS, and proteasome inhibition decreases mitochondrial tRNA import (32Brandina I. Smirnov A. Kolesnikova O. Entelis N. Krasheninnikov I.A. Martin R.P. Tarassov I. FEBS Lett. 2007; 581: 4248-4254Crossref PubMed Scopus (16) Google Scholar). Because mitoKARS appears to be poorly imported and aggregates in the presence of mutant SOD1, we tested whether the UPS is involved in degrading nonimported mitoKARS. Treatment with the proteasome inhibitor MG132 increases the unprocessed, nonimported, form of mitoKARS in cells co-expressing hSOD1 and mitoKARS (Fig. 4A, left panels), whereas cytoKARS content is unchanged (Fig. 4A, right panels). Because there is no evidence that mitochondria contain a prote" @default.
• W2024952866 created "2016-06-24" @default.
• W2024952866 creator A5021244706 @default.
• W2024952866 creator A5022025205 @default.
• W2024952866 creator A5032919938 @default.
• W2024952866 creator A5035747609 @default.
• W2024952866 creator A5068204379 @default.
• W2024952866 date "2008-10-01" @default.
• W2024952866 modified "2023-10-11" @default.
• W2024952866 title "Lysyl-tRNA Synthetase Is a Target for Mutant SOD1 Toxicity in Mitochondria" @default.
• W2024952866 cites W1480388053 @default.
• W2024952866 cites W1518241034 @default.
• W2024952866 cites W1555913203 @default.
• W2024952866 cites W179329376 @default.
• W2024952866 cites W1928433275 @default.
• W2024952866 cites W1965108432 @default.
• W2024952866 cites W1967495364 @default.
• W2024952866 cites W1970094644 @default.
• W2024952866 cites W1978372269 @default.
• W2024952866 cites W1978701238 @default.
• W2024952866 cites W1999899839 @default.
• W2024952866 cites W2014011193 @default.
• W2024952866 cites W2015266340 @default.
• W2024952866 cites W2022367303 @default.
• W2024952866 cites W2023568381 @default.
• W2024952866 cites W2038941603 @default.
• W2024952866 cites W2040724884 @default.
• W2024952866 cites W2052238208 @default.
• W2024952866 cites W2062900509 @default.
• W2024952866 cites W2066392481 @default.
• W2024952866 cites W2078040811 @default.
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• W2024952866 cites W2117212431 @default.
• W2024952866 cites W2125501179 @default.
• W2024952866 cites W2131226021 @default.
• W2024952866 cites W2134862369 @default.
• W2024952866 cites W2135118981 @default.
• W2024952866 cites W2135690573 @default.
• W2024952866 cites W2137368459 @default.
• W2024952866 cites W2137748756 @default.
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