Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate l-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

tetrasulfide (GSSSSG) with NEM and production of NEM adducts. GSSSSG (10 µM) was treated with NEM (1 or 10 mM) in 20 mM Tris-HCl or phosphate buffer (pH 5−9) at 37 °C for 1 h, followed by LC-ESI-MS/MS measurement of decomposed GSSSSG and NEM adducts. Data are means ± s.d. (n = 3). Whereas a simple disulfide (GSSG) is electrophilic, certain sulfur residues of GS-(S) n -SG were nucleophilic; nevertheless, all GS-(S) n -SG compounds tested decomposed in the reaction with various electrophiles. and different electrophilic compounds, e.g., p-chloromercuribenzoic acid (PCMB) (a), and application to the biotin-PEG 36 -MAL capture method, represented in b, for quantitative identification of endogenous polysulfidated proteins, which were isolated by reductive treatment of biotin-PEG 36 -MAL-bound avidin beads that captured polysulfidated proteins, followed by specific detection with Western blotting (b).    Fig. 1a. (b) Effects of NEM treatment of the amounts of CysS-(S) n -H detected in ADH5. Recombinant ADH5 (0.85 mg/ml) was alkylated with 6 mM HPE-IAM and 60 mM NEM at 37 °C for 5 min and was digested by 1 mg/ml Pronase, in 40 mM sodium acetate buffer (pH 5.5) in the presence of known amounts of isotope-labeled internal standards at 37 °C for 7 h, to produce cysteine or CysS-(S) n -H.
After addition of 0.1% formic acid and   spectra  are  presented  for  peptide  fragments containing carbamidomethyl-cysteine/cysteine polysulfide residues with their m/z values for ADH5 (a) and GAPDH (b). Polysulfidation occurred on 8 cysteine residues (of 15 cysteine residues of ADH5) (a) and 2 of 3 cysteine residues of GAPDH (b), which were analyzed by MS/Mascot.  (a) Schematic illustration of a new method for detection of polysulfide-bound nascent polypeptides, i.e., PUNCH-PsP, which we developed and successfully applied to specific identification of CysS-(S) n -H-containing polypeptides formed endogenously and present in the polypeptide exit tunnel of the ribosome. Because CysS-(S) n -H residues that are extruded and exposed outside the ribosome are readily alkylated with IAM or chemically modified, the CysS-(S) n -H residues of polypeptides that are newly synthesized and thus remain inside the polypeptide exit tunnel of the ribosome can be identified as the non-alkylated forms of the hydropolysulfides, as Fig. 1c shows. (b) Western blotting (streptavidin-peroxidase labeling) of nascent polypeptides de novo synthesized in ribosomes recovered from E. coli cells after transfection of the pGE30-hGAPDH-expressing vector used for this biotin-puromycin-avidin capture method as illustrated in a. Puromycin-labeled (biotinylated) nascent polypeptides were detected with ribosomes isolated from E. coli cells by Western blotting via the streptavidin-conjugated peroxidase reaction: 0.89 µg ribosomal protein with or without 5'-biotin-dC-puromycin treatment was applied to each lane. (c) The amino acid sequence of hGAPDH, with three cysteine residues being marked in red and the CysS-(S) n -H-containing peptide sequence identified by PUNCH-PsP, indicated by a red box. (d) MS spectra obtained from analysis of CysS-(S) n -H formation in nascent GAPDH, represented in Fig. 1c. (e) Direct identification by LC-Q-TOF of native forms of CysSH, CysSSH, and CySSSH residues present in mature GAPDH protein. Extracted-ion chromatograms and MS spectra are presented for peptide fragments containing cysteine/cysteine polysulfide residues with their m/z values for mature GAPDH. As soon as the recombinant GAPDH was isolated from E. coli, followed by quick digestion with trypsin, which was promptly subjected to the LC-ESI-Q-TOF analysis, in a similar manner as shown for the PUNCH-PsP method.  (a) Mitochondrial bioenergetics was analyzed by using JC-1 fluorescence imaging for WT and CARS2 KO HEK293T cells with or without transfection with WT or mutant CARS2. Fig. 8f illustrates the results of the morphometry obtained with this imaging analysis. Scale bars, 10 µm. (b) Assessment of mitochondrial electron flow in HEK293T CARS2 KO cells with or without adding back WT and C78/257D, K124/127A, and K317/320A mutants, as analyzed by measuring the oxygen consumption rate (OCR) with an extracellular flux analyzer. Time dependence of oxygen consumption before and after inhibition of mitochondrial respiration at complexes I and III by rotenone and antimycin A. Fig. 8g shows the result of the quantitative analysis of this OCR response. Data are means ± s.d. (n = 3).

L-[ 34 S]Cysteine was synthesized from
O-acetyl-L-serine and 34 S-labeled disodium sulfide via a unique catalytic reaction of cysteine synthase (CysK), whose recombinant protein was produced as described below. Briefly, O-acetyl-L-serine (20 mM) was reacted with 20 mM Na 2 34 S in 100 mM sodium phosphate buffer (pH 7.6) in the presence of 0.05 mg/ml CysK at 37 °C for 1 h. L-[ 34 S]Cysteine was purified from the reaction mixture by means of high-performance liquid chromatography (HPLC). The specific rat polyclonal antibody for mouse CARS2 and rabbit polyslonal antibody for CSE were produced by immunizing rats and rabbits with recombinant mouse CARS2 and rat CSE proteins, respectively, and by immunoaffinity purification, as described earlier 3 .
To generate pcDNA3-EGFP-mCARS1, the PCR products, after cleavage with HindIII and KpnI, were ligated into a pcDNA3 mammalian expression vector (Invitrogen). Open reading frames of CARSs from E. coli, cysteinyl-tRNA synthetase (EcCARS), with His tags at the N-termini (pCA24N-EcCARS), were obtained from the National BioResource Project at the National Institute of Genetics, Japan (NBRP-E.coli at NIG).
Construction of E. coli expression vectors PCR amplification of hADH5 cDNA was carried out with pCMV6-Entry-hADH5 as a template by using a primer set (Supplementary Table 1, restriction sites are underlined), and the resultant amplicon was digested with XhoI and cloned into the XhoI site of pET-15b to generate pET-15b-hADH5. To prepare a GAPDH expression vector, pCMV6-Entry-hGAPDH was used as a template for PCR amplification of hGAPDH cDNA with a primer set (Supplementary Table 1), and the amplicon was digested with NdeI and BamHI and cloned into the NdeI and BamHI sites of pET-30a(+) to generate pET-30a(+)-hGAPDH. To generate the vector pQE-70-hETHE1 expressing human ETHE1 (ethylmalonic encephalopathy 1; also known as GSSH dioxygenase), a cDNA library from A549 human lung cells corresponding to the ETHE1 gene (mitochondrial import signal truncated) was amplified by using a primer set (Supplementary Table 1). A resultant DNA fragment was cloned downstream of the T5 promoter between the SphI and BamHI sites of the pQE70 plasmid (Qiagen). pCMV6-Entry-hALDH1A1 was used as a template for PCR amplification of hALDH1A1 cDNA with a primer set (Supplementary Table 1). The resultant amplicon was digested with NdeI and XhoI and cloned into the NdeI and XhoI sites of pET-30a(+) to generate pET-30a(+)-hALDH1A1. To produce pCA24N-EcCARS K73A, pCA24N-EcCARS K76A, pCA24N-EcCARS K266A, pCA24N-EcCARS K269A, pCA24N-EcCARS C28S, pCA24N-EcCARS C209S, pCA24N-EcCARS C28D, pCA24N-EcCARS C209D, pCA24N-EcCARS K73/76A, pCA24N-EcCARS K266/269A, pCA24N-EcCARS C28/209S, and pCA24N-EcCARS C28/209D, site-directed mutagenesis was performed via inverse PCR with high-fidelity DNA polymerase KOD FX (Toyobo) and primer sets. pCA24N-EcCARS was used as a template for PCR amplification, and Supplementary Table 4 provides primer sets for introducing mutagenesis (mutant bases are underlined). The PCR protocol was as follows: denaturing, 94 ºC for 2 min; cycling (16 cycles), 98 ºC for 10 s, 55 ºC for 30 s, and 68 ºC for 1 min/kb. The PCR products were digested with SfiI (New England BioLabs, Ipswich, MA) and cloned into the SfiI site of pCA24N. To generate pET-15b-mCARS1, mCARS1 cDNA was PCR-amplified with pcDNA3-EGFP-mCARS1 as a template and a primer set (Supplementary Table 1). The resultant amplicon was digested with NdeI and cloned into the NdeI site of pET-15b to generate pET-15b-mCARS1. pCMV6-Entry-hCARS2 was used as a template for PCR amplification of human CARS2 cDNA, with a primer set (Supplementary Table 1), to obtain the amplicon, which was then digested with XhoI and cloned into the XhoI site of pET-15b to finally construct the pET-15b-hCARS2. To produce a CysK expression vector, Salmonella Typhimurium LT2 genomic DNA corresponding to CysK was obtained via PCR by using a primer set (Supplementary  Table 1), whose amplicon was inserted into the BamHI and SalI sites of pQE80L (Qiagen) to produce pQE80L-cysK. All plasmids generated by using PCR were verified via DNA sequencing.
Preparation and purification of recombinant proteins Recombinant hADH5 was purified according to a previously described method with modifications 5,6 . Briefly, E. coli BL21 (DE3) transformed with pET-15b-hADH5 was grown at 30 ºC to an OD 600 of 0.6-0.7 and induced by adding 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) (Sigma-Aldrich) for 18 h at 15 ºC. The other E. coli cultures (hETHE1, hGAPDH, hALDH1A1) were induced by adding 1 mM IPTG and culturing them for 3 h at 37 °C. The E. coli cells, transformed with various expression vectors, were harvested by centrifugation; resuspended in 20 mM Tris-HCl buffer (pH 8.0) containing 10 mM 2-mercaptoethanol (2-ME), 0.25 M NaCl, 1 mg/ml lysozyme, and protein inhibitor cocktail (Nacalai Tesque); and lysed by sonication. The resultant cell lysates were centrifuged at 10,000 × g for 20 min, and the supernatants were loaded onto a column packed with nickel nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) equilibrated in 20 mM Tris-HCl buffer (pH 8.0) containing 10 mM 2-ME and 0.25 M NaCl. The hADH5 protein was eluted with 200 mM imidazole and then dialyzed with 20 mM Tris-HCl (pH 8.0) containing 10 mM 2-ME, 10% glycerol, and 10 µM ZnSO 4 at 4 ºC for 18 h; the protein was stored at -80 ºC. Other proteins were purified in the same buffer but without 2-ME, glycerol, and ZnSO 4 . After desalting by means of the NAP-5 column with 20 mM Tris-HCl buffer (pH 8.0) and 1 mM Tris(2-carboxyethyl)phosphine (TCEP) (Nacalai Tesque), proteins were stored at -80ºC. To prepare a recombinant CysK, BL21 (DE3) pLysS was transformed with pQE80L-cysK and cultured for induction by 0.5 mM IPTG for 1 h at 30 °C, after which the CysK produced was purified by using Ni-NTA agarose and stored at -30 °C. Protein concentration was determined by using the Protein Assay CBB Solution (Nacalai Tesque), and protein purity was confirmed via SDS-PAGE.

Construction of mammalian expression vectors
To generate pPyCAGIP-FLAG-hADH5, the XhoI fragment of pET-15b-hADH5 was cloned into the XhoI site of pPyCAGIP-FLAG 4 . To generate pPyCAGIP-FLAG-hCARS2, the XhoI fragment of pET-15b-hCARS2 was cloned into the XhoI site of pPyCAGIP-FLAG. The same vectors containing various mutant hCARS2 genes were obtained via site-directed mutagenesis by using inverse PCR with high-fidelity DNA polymerase KOD FX (Toyobo) with pPyCAGIP-FLAG-hCARS2 as a template and primer sets for generation of pPyCAGIP-FLAG-hCARS2 C78/257D, K124/127A, and K317/320A. Supplementary Table 4 provides the primer sets for introducing mutagenesis (mutant bases are underlined).

Analysis of protein polysulfidation via gel shift assay
Various polysulfidated proteins were detected by using the biotin-PEG-MAL labeling gel shift assay (PMSA), which we recently developed 8,9 and herein describe as a modified version ( Supplementary Figs. 3a and 4). For example, purified recombinant proteins were applied to the PD SpinTrap G-25 column (GE Healthcare, Little Chalfont, England) equilibrated with RIPA buffer (10 mM Tris-HCl, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, pH 7.4) to remove reductants. After the proteins were quantified, they (0.3 mg/ml) were incubated in RIPA buffer that included 1 mM biotin-PEG 36 -MAL at 37 °C for 1 h. The mixture was subsequently incubated with various electrophiles (3 mM each), including IAM, MBB, 5'-dithiobis(2-nitrobenzoic acid) (DTNB), 4,4'-dithiopyridine (DTP), methyl methanethiosulfonate (MMTS), 2-methylsulfonyl benzothiazole (MSBT), 2-aminosulfonyl benzothiazole (ASBT), p-chloromercuribenzoic acid (PCMB), NEM, and 8-nitro-cGMP, at 37 °C for 1 h, after which the proteins were heat-denatured in the presence or absence of 5% 2-ME and subjected to SDS-PAGE and CBB staining. For PMSA analysis with lysates of A549 cells, Adh5 -/-MEFs expressing ADH5 and HEK293T cells were washed with ice-cold phosphate-buffered saline (PBS, pH 7.4) and collected with ice-cold RIPA buffer plus 1 mM TCEP and proteinase inhibitor cocktail (Nacalai Tesque). Cells were homogenized by 20 passages through a 26-gauge needle with a 1-ml syringe on ice and then centrifuged at 20,000 × g at 4 °C for 25 min. The supernatant was collected and incubated at 37 °C for 1 h in the presence of 1 mM TCEP, after which the protein was stored at -80 °C until use. After TCEP was removed with the PD SpinTrap G-25 column, cell lysate proteins (3 mg/ml) were analyzed according to the same procedure as that used for the PMSA assay for recombinant proteins, and individual proteins were detected by using Western blotting with specific antibodies.

Identification of polysulfidated proteins via the biotin-PEG-MAL capture method
PMSA can be applied to a biotin-PEG-MAL capture method ( Supplementary Fig. 3b) for quantitative identification of endogenous polysulfidated proteins, which are isolated by reductive treatment from the biotin-PEG-MAL-bound avidin beads, followed by specific detection with Western blotting. Briefly, cells were lysed with ice-cold RIPA buffer containing 1 mM biotin-PEG 36 -MAL and a proteinase inhibitor cocktail. After whole biotinylated proteins in the lysates were captured and enriched with Streptavidin Mag Sepharose (GE Healthcare), polysulfidated proteins were collected and subjected to Western blotting for Drp1. The biotin-PEG-MAL capture method is conceptually similar to the ProPerDP method recently reported 10 .

Preparation of mitochondria A mitochondrial fraction was isolated from WT and
Cars2 +/− mice as described previously 11 . Briefly, liver tissues were homogenized in isotonic buffer [10 mM HEPES, pH 7.4, 75 mM sucrose, 225 mM mannitol, 2 mM ethylenediaminetetraacetic acid (EDTA)] with a Teflon homogenizer for 15 strokes at 700 rpm and centrifuged at 700 × g for 10 min at 4 ºC. The supernatants were centrifuged again at 5,000 × g for 10 min at 4 ºC. The pellets were washed twice with the isotonic buffer. To examine the amount of CysSSH and cysteine released from mitochondria, the resupended mitochondria with the isotonic buffer were incubated at 37 °C for 1 h, followed by centrifugation (5,000 × g, 10 min, 4 ºC). The supernatants were alkylated with 1 mM HPE-IAM in RIPA buffer and were subjected to LC-ESI-MS/MS as mentioned above. To determine the amount of total CysSSH and cysteine generated by mitochondria, resupended mitochondria in isotonic buffer were incubated at 37 °C for 1 h. Mitochondria-containing mixtures were alkylated with 1 mM HPE-IAM in RIPA buffer and were subjected to LC-ESI-MS/MS as mentioned above.

Protein polysulfidation identified by LC-ESI-MS/MS and quadrupole -time-of-flight-MS
CysS-(S) n -H formed in various proteins were identified by means of LC-ESI-MS/MS analysis as just described and by LC-quadrupole (Q)-time-of-flight (TOF)-MS (LC-Q-TOF-MS) analysis combined with Mascot searches. Briefly, purified recombinant proteins were applied to a PD SpinTrap G-25 column equilibrated with 10 mM HEPES buffer (pH 7.5) to remove reductants, after which each protein (0.85 mg/ml) was alkylated with 6 mM HPE-IAM at 37 °C for 5 min and was digested by 1 mg/ml Pronase (Merck, Darmstadt, Germany), in 40 mM sodium acetate buffer (pH 5.5) in the presence of known amounts of isotope-labeled internal standards at 37 °C for 7 h, to produce cysteine or CysS-(S) n -H. After addition of 0.1% formic acid to the Pronase digest and centrifugation, the supernatants were subjected to LC-ESI-MS/MS. To identify the sites of polysulfur formation and sulfur numbers in each protein, recombinant hADH5 or hGAPDH, with 3 mM 2-ME or 0.3 mM TCEP, was alkylated with 10 mM IAM in 20 mM Tris-HCl buffer (pH 7.5) at 37 °C for 10 min, followed by digestion with 10 µg/ml trypsin (sequencing grade, modified; Promega) at 37 °C for 30 min. The digest samples diluted with 0.1% formic acid were subjected to LC-Q-TOF-MS as previously reported 2 . LC-Q-TOF-MS was performed with an Agilent 6510 Q-TOF mass spectrometer (Agilent Technologies), with an HPLC chip-MS system consisting of a nano pump with a four-channel microvacuum degasser, and a microfluidic chip cube. A microfluidic reverse-phase HPLC-chip (Zorbax 300SB-C18; 5-µm particle size, 75 µm inner diameter, and 43 mm length; Agilent Technologies) was used to separate the tryptic digest. The nano pump generated an isocratic flow of 400 nl/min with 0.1% formic acid and 3% acetonitrile. The capillary pump was for loading samples with a mobile phase of 0.1% formic acid at 4 µl/min. The ESI-Q-TOF instrument was operated in the positive ionization mode with an ionization voltage of 1750 V and a fragmentor voltage of 175 V at 300 °C. The selected m/z ranges were 300-1000 Da in the MS mode, and the instrument setting was 4 s -1 for the MS scan rate. The exact mass numbers of the peptide fragment containing the carbamidomethyl (CAM)-cysteine residues were obtained from Mascot MS/MS ion searches of the National Center for Biotechnology Information nonredundant (NCBI nr) database via the Matrix Science Web server Mascot version 2.2. Default search parameters were the following: enzyme, trypsin; maximum missed cleavage, 1; variable modifications, CAM (C); peptide tolerance, ±1.2 Da; MS/MS tolerance, ±1.2 Da. The digested peptide fragments with the polysulfidated cysteine residues were identified on the basis of their m/z values calculated by adding mass numbers of sulfur atoms equivalent to polysulfide chains comprising 1−4 sulfurs.

Identification of protein polysulfidation in cells and mouse tissues by LC-ESI-MS/MS
To identify protein polysulfidation produced in HEK293T cells and mouse liver, cell and tissues were homogenized with a Polytron homogenizer with RIPA buffer containing 5 mM HPE-IAM, followed by centrifugation (14,000 × g, 10 min, 4 °C). The addition of 5 mM HPE-IAM to the cell lysates and tissue peroxidase-conjugated secondary antibody (1:5000 dilution) for 1 h at room temperature. After the membranes were washed three times in TTBS, immunoreactive bands were detected via a chemiluminescence reagent (ECL Prime Western Blotting Detection Reagent; GE Healthcare) with a luminescent image analyzer (ImageQuant LAS 500; GE Healthcare). Densitometric analyses were performed to quantify these bands, with the signal intensity of the Western blotting images measured via ImageJ software. See Supplementary Figs. 16, 19, and 23 for uncropped blots.
Immunocytochemistry and transmission electron microscopy for assessment of mitochondrial morphology To investigate mitochondrial morphology under several experimental conditions with WT and CARS2 knockout (KO) cells, we performed immunocytochemistry with anti-TOMM20 (translocase of outer mitochondrial membrane 20) antibodies (No. Ab56783, Abcam) and anti-CARS2 antibody. Briefly, cultured WT or CARS2 KO HEK293T cells were plated in 8-well multichamber Millicell slides (Millipore) coated with polyethylene imine (PEI), with the cells being treated or untreated with various CARS2 vectors, and the slides were fixed with 4% paraformaldehyde solution at room temperature for 15 min. After PBS washes, cells were permeabilized with 0.5% Triton X-100 at room temperature for 10 min and washed with PBS. To block nonspecific antigenic sites, cells were incubated with 1% bovine serum albumin (BSA) (Sigma-Aldrich) at room temperature for 1 h. Cultured cells were then incubated at room temperature for 1 h with the primary antibodies (10 µg/ml) in PBS with 1% BSA, after which they were rinsed five times with PBS and incubated for 1 h at room temperature with Alexa Fluor 555 goat anti-mouse IgG (H+L) (No. A21424, Thermo Fisher Scientific, Rockland, IL) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) (No. A11034, Thermo Fisher Scientific) in PBS with 1% BSA. Cultured cells were washed with PBS, covered with ProLong Gold Antifade Reagent (Thermo Fisher Scientific), and examined with a Nikon EZ-C1 confocal laser microscope. Images were digitized and stored in PICT format by using a Color Chilled 3CCD Camera C5810 (Hamamatsu Photonics K.K., Shizuoka, Japan). We used ImageJ software for image processing and quantification. 4',6'-Diamindino-2-phenylindole (Thermo Fisher Scientific) served as a specific stain for the nucleus. Mitochondrial morphology was also examined by means of transmission electron microscopy (TEM), as described previously 15 . WT cells or CARS2 KO cells were plated in 6-well plates coated with PEI. CARS2 KO cells were transfected with WT flag-hCARS2 and various mutants. Cells were fixed with 2% glutaraldehyde/2% paraformaldehyde in PBS at room temperature for 15 min, followed by examination with a Hitachi H-7100S electron microscope.
Quantification of mtDNA mtDNA was quantified by using nuclear DNA (nDNA) content as a standard. Total genomic DNA including mtDNA was isolated by means of the QIAamp DNA Mini Kit (Qiagen). The relative abundance of mtDNA and nDNA in the total genomic DNA was quantified by qPCR with the CFX Connect Real-Time System (Bio-Rad Laboratories, Hercules, CA) and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories), with the Human Mitochondrial DNA (mtDNA) Monitoring Primer Set (Takara Bio). The following PCR protocol was used: 95 ºC for 4 min followed by 40 cycles each at 95 ºC for 15 s and 60 ºC for 30 s. The value of the threshold cycle number (Ct) of the mitochondrial genes and the nuclear genes was determined for each individual quantitative PCR run. The ΔCt [Ct (mitochondrial gene) -Ct (nuclear gene)] represents the relative abundance. The quantitative results were expressed as the copy number of mtDNA/cell by 2 Ct .

GTP-agarose pulldown assay
The GTP-agarose pulldown assay was performed according to the protocol of Gawlowski et al. with slight modification 16 . Briefly, cultured HEK293T cells with or without CARS2 expression were washed with ice-cold PBS and lysed in GTP-binding buffer (50 mM HEPES, 150 mM NaCl, 50 mM NaF, 1.5 mM MgCl 2 , 1 mM EGTA, 10% glycerol, and 1% Triton X-100, pH 7.4). The lysate was centrifuged (16,000 × g for 15 min at 4 ºC), and an aliquot of the supernatant (300 µg of protein) was incubated with 30 µl of GTP-agarose beads (Sigma-Aldrich) equilibrated in GTP-binding buffer for 1 h at room temperature. The beads were centrifuged (1,000 × g at 4 ºC for 1 min) and washed twice with GTP-binding buffer. The GTP-bound proteins were eluted with 2× Laemmli buffer with 20 mM DTT, and the supernatants were subjected to SDS-PAGE.
Separated proteins were electrotransferred to polyvinylidene fluoride membranes, and membrane-bound proteins were detected by using Western blotting with anti-Drp1 antibody as mentioned above.