Reagents and antibodies

Reagents: 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid (CDDO) (Cayman Chemical) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich), cycloheximide (Sigma-Aldrich), oligomycin (Sigma-Aldrich), MKT-077 (Sigma-Aldrich). Antibodies for Western blot analysis: LONP1 (Proteintech, 1:3000), OXA1L (Proteintech 1:10000), mtHSP70 (Proteintech, 1:10000), GRPEL1 (Proteintech, 1:5000), TIM44 (Proteintech, 1:5000), CLPX (Proteintech, 1:3000), VDAC1 (Abcam, 1:3000), NDUFA9 (Abcam, 1:3000), DNAJA3 (Santa Cruz BioTech, 1:1000), HSP60 (Santa Cruz BioTech, 1:3000), TOM20 (Santa Cruz BioTech, 1:3000), anti-FLAG M2 (Sigma-Aldrich, 1:10000), anti-HA.11 (Covance, 1:5000).

Cell culture

143B, HeLa, U2OS and LONP1 patient cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 μg/ml uridine, 2 mM pyruvate and 2 mM glutamine. For glucose or galactose culture, the base DMEM solution lacked glucose, pyruvate, and glutamine (Invitrogen, A14430) and was supplemented with 10% dialyzed FBS, 2 mM glutamine, and 10 mM glucose or galactose.

Doxycycline-inducible expression

The Retro-X™ Tet-On^® Advanced system (Clontech) was used to establish 143B cells with doxycycline-inducible expression of LONP1-FLAG, OXA1L-FLAG and mtHSP70-HA. Cells were incubated with doxycycline (0.1 μg/ml for OXA1L-FLAG and mtHSP70-HA; as indicated concentration for LONP1-FLAG) to induce protein expression.

Triton X-100 extraction

Cells were detached with trypsin, pelleted, and lysed with Triton X-100 buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) containing HALT protease inhibitors (Thermo-Pierce). Cell lysates were centrifuged at 20,817g for 10 min at 4 °C to separate into supernatant and pellet fractions. The supernatant and insoluble pellet were reconstituted in equivalent volumes of sample buffer and analyzed by SDS-PAGE and immunoblotting.

Plasmid constructs and mutagenesis

OXA1L-FLAG, LONP1-FLAG and mtHSP70-HA were expressed from the mammalian vectors pQCXIP (Clontech) or pRetro-Tight-Pur (Clontech). Vectors were digested with NotI and AgeI (New England Biolabs) and ligated with the following fragments: the LONP1 gene, PCR-amplified from the pcDNA-LONP1 vector (kindly provided by Carolyn Suzuki, Rutgers New Jersey Medical School); the OXA1L or mtHSP70 genes, PCR-amplified from a human cDNA library. PCR primers were designed to add a FLAG or HA tag at the C-terminus. Point mutations were introduced into the coding sequence of LONP1 with overlap extension PCR-based mutagenesis.

For in vitro expression of OXA1L and CLPX, the DHFR control vector included in the PURExpress kit (New England Biolabs) was digested with NdeI and NotI or BamHI (New England Biolabs), and ligated with the OXA1L (amino acid residues 72–435) gene and CLPX (amino acid residues 57–633) gene, PCR-amplified from a human cDNA library.

For bacterial expression of DNAJA3, pETDUET-1 (Novagen) was digested with BamHI and NotI (New England Biolabs), and ligated with the short isoform of the DNAJA3 (amino acid residues 66–453) gene, PCR-amplified from a human cDNA library. All primers used for the cloning and LONP1 mutagenesis are listed in Supplementary Table 1.

Recombinant protein expression and purification

Recombinant proteins were expressed in Rosetta (DE3) BL21 Escherichia coli (EMD Millipore).

For His-tagged mtHSP70 expression, pET28a-mtHSP70 and pET23a-HEP1 (kindly provided by Júlio César Borges, University of São Paulo) were co-transformed into bacteria¹. For His-tagged GRPEL1, LONP1 and DNAJA3 expression, pET-His6-GRPEL1 (kindly provided by Henna Tyynismaa, University of Helsinki), pPROEX-LONP1 (kindly provided by Carolyn Suzuki, Rutgers New Jersey Medical School) and pETDUET-1-DNAJA3 vectors were transformed into bacteria. Transformed bacteria were grown in 50 ml terrific broth containing 25 μg/ml chloramphenicol and either 100 μg/ml ampicillin or 50 μg/ml kanamycin at 37 °C to an OD₆₀₀ of 1.2, shifted to 16 °C (for LONP1, mtHSP70 and DNAJA3) or 30 °C (for GRPEL1), and induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside for 18 h. Cells were harvested by centrifugation at 3500 rpm for 30 min with a swinging bucket rotor (BioFLex HC, Thermo Fisher), and pellets were stored at −20 °C. For purification, bacterial pellets were thawed and resuspended in buffer (25 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10% glycerol, and HALT protease inhibitors (Thermo-Pierce)), lysed by sonication, and cleared by centrifugation at 50,000g for 30 min at 4 °C in Oakridge tubes in the A27-8 × 50 rotor (Thermo Fisher). The soluble supernatant was incubated with washed TALON beads (Clontech) for 4 h at 4 °C. After extensive washing of the beads with buffer (25 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10% glycerol, 20 mM imidazole), proteins were eluted from TALON beads with elution buffer (25 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10% glycerol, 250 mM imidazole). The eluted proteins were further concentrated in buffer (25 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10% glycerol, 25 mM imidazole) by Amicon Ultra Centrifugal Filters (Millipore). Proteins were flash frozen and stored at −80 °C.

Isolation of mitochondria

Mitochondria were isolated from cells by differential centrifugation. Cells were collected by scraping in isolation buffer (220 mM mannitol, 70 mM sucrose, 80 mM KCl, 1 mM MgCl₂, 0.5 mM EDTA, 10 mM K + HEPES, pH7.4, and HALT protease inhibitors) and Dounce homogenized (25 strokes) on ice. Cell debris and nuclei were removed by centrifugation twice at 650g for 5 min at 4 °C. A crude mitochondrial fraction was pelleted by centrifugation at 10,000g for 10 min at 4 °C and washed once in isolation buffer.

RNA interference

For shRNA-mediated stable knockdown of LONP1 or DNAJA3, the indicated cell lines were infected with retrovirus expressing shRNA from the human H1 promoter. The targeted sequences were: LONP1-Sh (5′-GGGACATCATTGCCTTGAACC-3′), DNAJA3-sh (5′-GCTGTTCAGGAAGATCTTTGG-3′), CLPP- sh (5′-GCTCAAGAAGCAGCTCTATAA-3′), CLPX- sh (5′-GGAGTATGACTCTGGAGTTGA-3′), non-target shRNA (5′-CGTTAATCGCGTATAATACGC-3′). For LONP1 small interfering RNA (siRNA) experiments, dicer-substrate RNA (DsiRNA) duplexes (5′-ATCATTGCCTTGAACCCTCTCTACA-3′) were purchased from Integrated DNA Technologies (IDT). The following predesigned DsiRNAs were purchased from IDT: negative control DsiRNA, hs.Ri.DNAJA3.13.2, hs.Ri.HSPA9.13.2, hs.Ri.HSPD1.13.1, hs.Ri.GRPEL1.13.3 and hs.Ri.TIMM44.13.2. Transfections of siRNA were performed using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s protocol.

In vitro translation of OXA1L and CLPX

In vitro translation of OXA1L and CLPX was performed using the PURExpress^® In Vitro Protein Synthesis Kit (New England Biolabs). Reactions for OXA1L and CLPX expression were incubated in the absence or presence of recombinant LONP1 and mtHSP70 chaperone components for 8 h at 37 °C and ultracentrifuged at 186,000g for 1 h at 4 °C to separate into supernatant and pellet fractions.

Immunostaining and Imaging

Cells were fixed in 10% formaldehyde, permeabilized in 0.1% Triton X-100, and immunostained with antibody against FLAG M2 (Sigma, 1:500), HA.11 (Covance, 1:500), TOM20 (Santa Cruz BioTech, 1:500) or HSP60 (Santa Cruz BioTech, 1:500). Immunofluorescent images were obtained with a Zeiss LSM 710 confocal microscope (Carl Zeiss). Image data were analyzed using ImageJ (v. 1.51J8).

Intracellular ATP measurement

The ATP Determination Kit (Thermo Fisher Scientific) was used for detection of intracellular ATP in cell lysates. Cells were resuspended in 0.1 M Tris-HCl, pH 7.4 and lysed by sonication with three cycles of 10 sec on and 30 s off at 20% amplitude (MISONIX S-4000 sonicator). Cell lysates were used for intracellular ATP measurement. Values were normalized to protein concentrations of the lysates. Results were collected from three independent cultures for each sample. Data are presented as means ± standard deviation.

Co-immunoprecipitation

Trypsinized cells expressing FLAG-tagged mtHSP70 or LONP1 were lysed in immunoprecipitation (IP) buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 5% glycerol, 1% Igepal CA-630) plus HALT protease inhibitors (Thermo-Pierce). The cell lysate was cleared by centrifugation at 20,817g for 10 min at 4 °C, and the supernatant was mixed with ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich) for 30 min at 4 °C. For immunoprecipitation of in vitro translated OXA1L and CLPX, the reaction was cleared by ultracentrifugation at 186,000g for 1 h at 4 °C and the supernatant was incubated with anti-OXA1L antibody (Proteintech) or anti-LONP1 antibody (Proteintech) for 2 h at 4 °C. Protein A/G-agarose (Thermo-Pierce) was subsequently added and incubated for additional 1 h at 4 °C to capture the immuno-complexes. After the beads were washed three times with IP buffer, bound proteins were eluted by boiling in SDS-PAGE sample buffer and analyzed by SDS-PAGE and immunoblotting. Input samples were collected prior to mixing with beads and 10% (for cell lysates) or 20% (for in vitro translation reactions) of the input samples were used for immunoblot analysis.

Mass spectrometry analyses of mitochondrial protein aggregates

Mitochondria were isolated in biological triplicates from control, LONP1 or DNAJA3 knockdown, and CDDO ± cycloheximide (CHX)-treated 143B cells and lysed using Triton X-100 extraction. Insoluble mitochondrial protein pellets were separated by centrifugation at 20,817g for 10 min at 4 °C and solubilized in urea buffer (8 M urea, 100 mM Tris-HCl, pH 8.5). The solubilized samples were digested by trypsin overnight at room temperature and desalted by HPLC with a Microm Bioresources C8 peptide Microtrap. Subsequently, the samples were subjected to LC-MS/MS analysis on a nanoflow LC system, EASY-nLC 1200, (Thermo Fisher Scientific) coupled to a Q Exactive HF Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a Nanospray Flex ion source. Samples were directly loaded onto a PicoFrit column (New Objective, Woburn, MA) packed in-house with ReproSil-Pur C18AQ 1.9 μm resin (120 Å pore size, Dr. Maisch, Ammerbuch, Germany) heated to 60 °C or an Aurora 25 cm × 75 µm ID, 1.6 µm C18 column (Ion Opticks, Victoria, Australia) heated to 50 °C. The peptides were separated with a 60 min gradient at a flow rate of 220 nL/min for the in-house packed column or 350 nL/min for the Aurora column. The gradient was as follows: 2–6% Solvent B (3.5 min), 6–25% B (41.5 min), and 25–40% B (15 min) and to 100% B (10 min). Solvent A consisted of 97.8% H₂O, 2% ACN, and 0.2% formic acid and solvent B consisted of 19.8% H₂O, 80% ACN, and 0.2% formic acid. The Q Exactive HF Orbitrap was operated in data dependent mode with the Tune (version 2.7 SP1build 2659) instrument control software. Spray voltage was set to 2.5 kV, S-lens RF level at 50, and heated capillary at 275 °C. Full scan resolution was set to 60,000 at m/z 200. Full scan target was 3 × 10⁶ with a maximum injection time of 15 ms. Mass range was set to 400–1650 m/z. For data dependent MS2 scans collected at 30,000 resolution the loop count was 12, target value was set at 1 × 10⁵, and intensity threshold was kept at 1 × 10⁵. Isolation width was set at 1.2 m/z and a fixed first mass of 100 was used. Normalized collision energy was set at 28. Peptide match was set to off, and isotope exclusion was on. Data acquisition was controlled by Xcalibur (4.0.27.13) and all data were acquired in profile mode.

Data analysis

Raw data were searched in Proteome Discoverer 2.4 (Thermo Scientific) using the Byonic search algorithm (Protein Metrics) and Uniprot human database. PD-Byonic search parameters were as follows: fully Tryptic peptides with no more than 2 missed cleavages, precursor mass tolerance of 10 ppm and fragment mass tolerance of 20 ppm, and a maximum of 2 common modifications and 2 rare modifications. Cysteine carbamidomethylation was set as a static modification, while methionine oxidation was a common dynamic modification (up to 2 per peptide). Methionine loss on protein N-termini, methionine loss + acetylation on protein N-termini, protein N-terminal acetylation, lysine acetylation, and phosphorylation of serine, threonine, and tyrosine were rare dynamic modifications (only 1 each). Byonic protein-level FDR was set at 0.01, while Percolator FDRs were set at 0.01 (strict) and 0.05 (relaxed). In the consensus step, peptide and PSM FDRs were set at 0.001 (strict) and 0.01 (relaxed), with peptide confidence at least medium, lower confidence peptides excluded, minimum peptide length set at 6, and apply strict parsimony set to false.

LFQ was then performed with the Minora feature detector, feature mapper, and precursor ions quantifier nodes. Retention time alignment was performed with maximum RT shift of 5 min and a minimum S/N threshold of 10. Quantified peptides included unique + razor, protein groups were considered for peptide uniqueness, shared Quan results were not used, Quan results with missing values were not rejected, and precursor abundance was based on intensity. Abundances were normalized using the total intensities of all non-mitochondrial proteins identified in a pre-search of the raw data files from the control, LONP1 kd, DNAJA3 kd and CDDO ± CHX samples with identical search parameters. Imputation was then performed using the low abundance resampling method.

Statistical analysis of protein abundances was performed using methods developed for linear models for microarray data³⁵ available as a package in R (R version 4.0.2, limma version 3.44.3). Samples were normalized between arrays, modeled for expression using the linear model fit, and fold changes were calculated as a log2 function, and multiple testing correction utilized the Benjamini-Hochberg method to calculate FDR-adjusted p-values³⁶. Proteins and peptides identified are listed in Supplementary Data 3 and 4. Proteins were then annotated as being localized to the mitochondria based on annotation in either MitoCarta or UniProt, and only those mitochondrial proteins with 2 or more unique peptides and quantified in at least 5 of the 9 replicates from the LONP1 kd, DNAJA3 kd, and CDDO samples were subsequently retained (Supplementary Data 1) for further analysis and included in figure generation. Proteins considered significant were enriched at least 2-fold over control with a BH-adjusted p-value < 0.05. Mass spectrometry data files have been deposited at ProteomeXchange, with access code PXD021939.

Statistics and reproducibility

For Western blotting, confocal microscopy, ATP measurement, and mass spectrometry, all experiments were independently repeated 2 or 3 times.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.