Human HEK-293T and HeLa cells were obtained from ATCC and grown in full medium: DMEM and EMEM, respectively, supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco) at 37 ºC and 5% CO2.
Construction of plasmids and plasmid transfection
For the generation of pcDNA ELP3 and pcDNA mt-ELP3, the corresponding ORFs were amplified by PCR and cloned into a pCDNA4.T0 vector with a Flag tag at the N terminus.
The KAT domain mutant of ELP3 (pcDNA mt-ELP3 mut) was generated by PCR-based mutagenesis using specific primers harboring the desired mutation and pcDNA mt-ELP3 was used as a template. The mutation was confirmed by sequencing (Figure S4). The primers used for generation of this mutant were as follows:
KAT-F, 5′-CAAACTGATGCTGAAATTTAGTAGG -3′;
For GTPBP3 plasmid, GTPBP3 (NM_001128855) Human Tagged ORF Clone was purchased from Origene (cat # RC225798).
For the transfection assay, cells were transiently transfected with control siRNA duplex (OriGene #SR30002) and two Stealth siRNAs targeting ELP3 (ELP3 siRNA1(OriGene#SR310519A) and ELP3 siRNA 2 (OriGene#SR310519B)) or with pcDNA 4/T0 derivative plasmid expressing canonical ELP3 (pcDNA 4/T0-ELP3), mt-ELP3 (pcDNA 4/T0-mt-ELP3), mutant mt-ELP3 (pcDNA 4/T0-mt-ELP3) or empty pcDNA 4/T0 (pcDNA 4/T0) with Lipofectamine 3000 reagent, according to the manufacturer’s instructions. The cells were processed after 2 days of transfection.
Generation of inducible HEK293 Cell Lines
Inducible HEK293 cell lines expressing mt-ELP3 were generated by using the Flp-In T-REx Core Kit from Invitrogen. Briefly, the cDNA encoding the mt-ELP3 was subcloned into the inducible expression vector, pcDNA5/FRT/TO using pcDNA mt-ELP3 as a template. Flp-In T-REx-293 cells were then co-transfected with the inducible expression vector pcDNA5/FRT/TO containing the mt-ELP3 cDNA and the pOG44 vector encoding the Flp recombinase by using OptiMEM/Lipofectamine2000 according to the manufacturer’s instructions. After 48h of transfection, cells were washed with PBS and incubated with selective medium containing 200 mg/ml hygromycin. The selective medium was changed at regular intervals until the desired number of cells was grown. Thereafter, the hygromycin-resistant cells were maintained in DMEM containing 10% FBS, 5ug/ml Blasticidin and 250ug/ml zeocin.
The expression of mt-ELP3 was induced by the addition of tetracycline (1ug/ml) to the culture medium for 24h.
RNA isolation and APM-northern blotting analysis
Total RNA isolation was carried out using Trizol reagent (Invitrogen), according to the manufacturer’s recommendations. Northern blotting of APM-containing gels was performed as described to assess the thiolation status of tRNAs (Meseguer et al., 2015). Briefly, total RNA (5 μg) was run on a 15% polyacrylamide gel containing 7M urea and 10 μg/ml APM and then transferred to positively charged nylon membranes (Roche). Pre-hybridization and hybridization steps were performed with Dig Easy Hyb (Roche), according to the manufacturer’s instructions. The presence of tRNA species was detected with a specific DIG-labeled synthetic oligodeoxynucleotide probes (Table S1).
In vitro angiogenin assay
Angiogenin nuclease-sensitivity assays were performed essentially as described (Rachid Boutoual et al., 2018). In brief, 1 μg of total RNA were mixed with 2.5 μg/ml recombinant angiogenin (ANG) in buffer (30 mM HEPES, pH 7.4, 30 mM NaCl, 0.01% BSA). Mixtures were incubated at 37˚C for the indicated times. Angiogenin was inactivated by adding 5 μl of Gel Loading Buffer II (Life Technologies). The digested RNA samples were separated on 15% polyacrylamide, 8M urea gels and transferred to positively charged nylon membranes. The RNA was cross-linked to the membrane (60˚C, 1 h) using freshly prepared EDC cross-linking solution (Kim et al., 2010)(Pall et al., 2007). Pre-hybridization and hybridization were performed with Dig Easy Hyb (Roche), according to the manufacturer’s instructions. tRNA species were detected with specific DIG-labeled synthetic oligodeoxynucleotide probes (Table S1).
Blue native electrophoresis analysis
BN-PAGE was carried out as detailed (Sasarman et al., 2011). Samples containing 15 μg of protein were separated on 3–12% Bis-Tris Novex NativePAGE gel (Life Technologies). The relative levels of the assembled respiratory complexes I-V were assessed by western blot with following antibodies: anti-ND1(Thermo Fisher, 19703-1-AP),anti-SDHA (Cell signaling, 5839),anti-MTCO1(Thermo Fisher, 459600), anti-ATP5a (Abcam, 11448) and anti-Cytb (Abclonal, A17966).Horseradish-peroxidase-conjugated anti-rabbit IgG (Sigma, A6154) and anti-mouse IgG (Sigma, A4416) antibodies were used as secondary antibodies and protein signals were detected using the ECL system. Bis-Tris Novex NativePAGE (3–12%) gels were stained using Coomassie protein staining solution (50% methanol, 7% acetic acid and 0.1% Coomassie Brilliant Blue R stain).
Western blots and subcellular fractionation
HEK293T cells were collected by centrifuging at 500xg for 5 min. Cells were lysed in RIPA buffer, and the homogenates were cleared by centrifugation and analyzed for protein concentration using BCA kit (Thermo Scientific). Protein lysates were separated on 4-20% gradient gels and transferred to PVDF membranes.
Subcellular fractionation was carried out as described with a slight modification (Park et al., 2013). Cells were gently homogenized in hypotonic buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA) containing protease inhibitors (0.5 mM PMSF and protease inhibitor complete (Roche)) for 30 min on ice with the use of a dounce homogenizer until the vast majority of cells were ruptured and the nuclei were stained by Trypan blue, and then centrifuged at 800xg for 5 min at 4°C to obtain a pellet (nuclear fraction). The supernatant was centrifuged at 15,000xg for 10 min at 4°C to obtain a mitochondrial fraction (pellet) and cytosolic fraction (supernatant). The mitochondrial pellet was washed and suspended in hypotonic buffer. Protein concentration and immunoblotting were done as described above. Where indicated, the mitochondrial fraction was incubated with proteinase K (0.5–2 mg/ml) in hypotonic buffer in the presence of increasing concentrations of digitonin or proteinase K with 0.3% Triton X-100 for 15 min at 37 °C. After 15 min, the digestion was ended by adding 5 μL of 200 mM PMSF. The samples were boiled for 5 min and analyzed by western blotting.
For immunodetection, the following antibodies were used: anti-ELP3 (cell signaling, 5728S), anti-COX IV (cell signaling, 4850T), anti-GAPDH (Abcam, AB9484), anti-HSP60 (cell signaling, 4870S), anti-TOM20 (cell signaling, 42406), anti-Flag (Sigma, F1804-200UG), anti-acetylated-lysine (cell signaling, 9441) and anti-VDAC (cell signaling,4661).
HeLa cells were cultured on coverslips in 24-well plates overnight. After transient transfections, cells were washed with PBS, fixed with 4% paraformaldehyde–PBS for 20min at RT, washed with PBS, permeabilized with 0.3% Triton X-100 in PBS for 15 min and washed again with PBS. After being blocked with a solution containing 2% BSA, 0.05% Triton X-100 in PBS for 30 min at room temperature (RT), cells were incubated with anti-COX IV and anti-Flag antibodies in blocking solution for 1 h at RT. Upon washing with blocking solution, bound antibodies were subsequently detected by incubation, as appropriate, AlexaFluor 594-conjugated anti-rabbit (11012, Invitrogen) and AlexaFluor 488-conjugated anti-mouse (A11001, Invitrogen) secondary antibodies in blocking solution for 1h at RT. After staining with 1 mg/mL DAPI in PBS, cells were examined by using a Leica confocal microscope.
293HEKT cells were co-transfected with indicated plasmids. After 72h post-transfection, transfected cells were harvested and lysed with IP lysis buffer (thermos scientific). The supernatants were obtained by centrifugation at 16,000 × g for 10 min at 4°C. Coimmunoprecipitation was performed using Dynabeads Protein G immunoprecipitation kit (Life Technologies) following the manufacturer's instructions. Briefly, 10µg of anti-Flag M2 antibody was incubated with protein G Dynabeads for 10 minutes at RT. After washing, the antibody-Dynabeads complex was incubated (1 hour) with 300µg protein lysates (supernatant). Following washes, immune complexes were eluted in 20µl elution buffer and analyzed by immunoblotting. Dynabeads were also incubated with anti-Flag M2 antibody (without lysates) and with lysates (without anti-Flag M2 antibody) and were used as negative control.
De novo mitochondrial protein synthesis
De novo mitochondrial protein synthesis was performed as described (Zorkau et al., 2020). HEK239T cells were seeded in lab-tek chamber slides (ThermoFisher Scientific) and transfected with pcDNA-T0 or Flag-tagged pcDNA-mt-ELP3. After transfection, cells were incubated in methionine-free DMEM for 30 min. Then, the medium was replaced by methionine-free DMEM containing the methionine analogue L-homopropargylglycine (HPG), and cytosolic and mitochondrial translation were inhibited with 50 μg/ml cycloheximide (CHX) and 100 μg/ml chloramphenicol (CHL), respectively. After labelling period, slides were immediately placed on ice, and cells were pre-permeabilized with 0.005% digitonin in buffer A (10 mM HEPES/KOH, 10 mM NaCl, 5 mM MgCl2 and 300 mM sucrose in H20, pH 7.5) for 2 min at RT before being fixed in pre-warmed 8% formaldehyde in buffer A for 15 min. Then, cells were fully permeabilized with 0.5% Triton X-100 in PBS. Pulse-labelled mitochondrial proteins were detected with a copper-catalyzed azide–alkyne cycloaddition (600 mM copper sulphate, 1.2 mM BTTAA, 40 μM picolyl Alexa Fluor 594 (CLK-1296-1) azide and 2 mM sodium ascorbate in PBS) by a click chemistry reaction for 1h at RT.
Samples were imaged using LSM700 confocal microscope. After imaging, each HPG-stained cell was selected by drawing an outline near the visible edge. The mean pixel intensity was then calculated for each bounded region representing the cell. Selection and analysis were performed with custom software written in Python.
For fluorescence intensity measured by flow cytometry, Brilliant Violet 421™ anti-DYKDDDDK (Flag-Tag) antibody (Cat# 637322, BioLegend) at 1:50 dilution was also added to the click reaction to sort transfected cells expressing mt-ELP3. Cells were then washed with 1XPBS and resuspended in 100µl of 1xPBS to acquire on Flow cytometer. Cells were acquired on FACS Aria II (BD Biosciences) with HPG signal collected in the Cherry filter while the Flag was collected in BV421 filter. Post-acquisition fcs files were analyzed using Flow jo v10 software (BD Biosciences). Cells were plotted as bimodal distribution of Cherry (HPG) Vs BV421 (Flag), MFI (mean fluorescence intensity) of HPG was quantified on Flag+ and Flag- population.
Seahorse XF assays
Oxygen consumption rate (OCR) was assessed by a Seahorse XF96 Analyzer.
Cells were seeded in Seahorse XF96 microplates at 4x104 cells/well and incubated at
37°C in a CO2-free incubator for 1 h in prepared medium (1.8 mM CaCL2,139 mM NaCl, 20 mM HEPES, 1 mM NaHCO3, 25 mM glucose,1 mM pyruvate and 4 mM glutamine, pH 7.4) prior to analysis. Bioenergetic profiling was performed by monitoring oxygen consumption at basal levels, followed by the sequential injection of the following inhibitors: 2 μM oligomycin, 0.5 μM carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone, 4 μM antimycin and 4 μM myxothiazol. All measurements were normalized against total number of cells in each well via imaging stained nuclei.
1) Protein digestion, acylation enrichment and desalting
Three independent biological experiments (with additional technical replicates) of HEK 293T cells control cells (-Tetra) and cells overexpressing mitochondrial ELP3 (+Tetra) were investigated by acetyl enrichment followed by mass spectrometric analysis. Cell lysates were immersed in lysis buffer containing 8 M urea, 200 mM triethylammonium bicarbonate (TEAB), pH 8.5, 75 mM sodium chloride, 1 μM trichostatin A, 3 mM nicotinamide, and 1x protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA), and homogenized for 2 cycles with a Bead Beater TissueLyser II (Qiagen, Germantown, MD) at 24 Hz for 3 min each. Lysates were clarified by spinning at 15,700 x g for 15 min at 4°C, and the supernatant containing the soluble proteins was collected. Protein concentrations were determined using a bicinchoninic acid protein (BCA) Assay (Thermo Fisher Scientific, Waltham, MA), and subsequently 1-2 mg of protein from each sample were brought to an equal volume using a solution of 8 M urea in 50 mM TEAB, pH 8. Proteins were reduced using 20 mM dithiothreitol (DTT) in 50 mM TEAB for 30 min at 37 °C, and after cooling to room temperature, alkylated with 40 mM iodoacetamide (IAA) in 50 mM TEAB for 30 min at room temperature in the dark. Samples were diluted 4-fold with 50 mM TEAB, pH 7.5, and proteins were digested overnight with a solution of sequencing-grade trypsin (Promega, San Luis Obispo, CA) in 50 mM TEAB at a 1:50 (wt:wt) enzyme:protein ratio at 37°C. This reaction was quenched with 1% formic acid (FA) and the sample was clarified by centrifugation at 2,000 x g for 10 min at room temperature. Clarified peptide samples were desalted with Oasis 10-mg Sorbent Cartridges (Waters, Milford, MA), and subsequently all desalted samples were vacuum dried. The digestions were re-suspended in 1.4 mL of immunoaffinity purification (IAP) buffer (Cell Signaling Technology, Danvers, MA) containing 50 mM 4-morpholinepropanesulfonic acid (MOPS)/sodium hydroxide, pH 7.2, 10 mM disodium phosphate, and 50 mM sodium chloride for PTM enrichment. Peptides were enriched for acetylation with anti-acetyl antibody conjugated to agarose beads (Acetyl-Lysine Motif Kit; Cell Signaling Technology, Danvers, MA). This process was performed according to the manufacturer protocol, and each sample was incubated overnight with half a vial of washed beads. Finally, acetyl-enriched peptides were eluted from the antibody-bead conjugates with 0.1% trifluoroacetic acid in water and were desalted using C-18 zip-tips (Millipore, Billerica, MA). Samples were vacuum dried and re-suspended in 0.2% FA in water.
2) Mass Spectrometric Analysis
Samples were analyzed by reverse-phase HPLC-ESI-MS/MS using an Eksigent Ultra Plus nano-LC 2D HPLC system (Dublin, CA) with a cHiPLC system (Eksigent) which was directly connected to a quadrupole time-of-flight (QqTOF) TripleTOF 6600 (or TripleTOF 5600) mass spectrometer (SCIEX, Concord, CAN). After injection, peptide mixtures were loaded onto a C18 pre-column chip (200 µm x 0.4 mm ChromXP C18-CL chip, 3 µm, 120 Å, SCIEX) and washed at 1-2 µl/min for 10 min with the loading solvent (H2O/0.1% formic acid) for desalting. Subsequently, peptides were transferred to the 75 µm x 15 cm ChromXP C18-CL chip, 3 µm, 120 Å, (SCIEX), and eluted at a flow rate of 300 nL/min with 2-3 hour gradients using aqueous (A) and acetonitrile (B) solvent buffers.
3) Data-dependent acquisition (DDA) to identify acetylated peptides and build spectral libraries. Every cycle consisted of one 250 ms precursor ion scan followed by isolating the top 30 most abundant precursor ions between 400-1,500 m/z (tandem mass spectra accumulation time of 100 ms yielding a total cycle time of 3.25 sec; ‘high sensitivity’ product ion scan mode, software: Analyst 1.7; build 96) as previously described (Schilling et al., 2015).
4) Data-independent acquisition (DIA) to quantify the PTM enriched peptides using the TripleTOF 6600 mass spectrometer. Every cycle consisted of one 250 ms precursor ion scan with 400-1,250 m/z mass range. Subsequently, windows of variable width are passed in incremental steps over the full mass range (m/z 400-1,250). The cycle time of 3.2 sec includes the 250 msec precursor ion scan followed by 45 msec accumulation time for each of the variable 64 DIA segments (variable windows isolation scheme (Schilling et al., 2017)) monitoring fragment ion masses between 100-1,500 m/z.
5) Mass-spectrometric data processing and quantification. Mass spectrometric data-dependent acquisitions (DDA) were analyzed using the database search engine ProteinPilot (SCIEX Beta 4.5, revision 1656) using the Paragon algorithm (18.104.22.168,1654). Using these database search engine results MS/MS spectral libraries were generated. Quantification was performed in Skyline using MS1 Filtering and DIA/SWATH MS2 data quantification (using XICs of 6-10 MS/MS fragment ions, typically y- and b-ions, matching to specific peptides present in the spectral libraries) as previously described (Schilling et al., 2012)(Rardin et al., 2015)(Gut et al., 2020).
6) Data availability. The mass spectrometric raw data are deposited at ftp://massive.ucsd.edu with the MassIVE ID MSV000088668 (password winter): Go to
Enter the username and password in the upper right corner of the page:
Username: MSV000088668_reviewer; Password: winter
The raw data is also available at ProteomeX change with the ID PXD030850 (will be public upon release).
The statistical analyses were performed using Graph Pad Prism 9. Student’s t-test was used in all comparisons of data. The statistically significant differences between the means were indicated by asterisks (*p < 0.05, **p < 0.01 or ***p < 0.001), and non-significant differences by n.s.