1. Biochemistry and Chemical Biology
Download icon

Mitochondrial fatty acid synthesis coordinates oxidative metabolism in mammalian mitochondria

  1. Sara M Nowinski
  2. Ashley Solmonson
  3. Scott F Rusin
  4. J Alan Maschek
  5. Claire L Bensard
  6. Sarah Fogarty
  7. Mi-Young Jeong
  8. Sandra Lettlova
  9. Jordan A Berg
  10. Jeffrey T Morgan
  11. Yeyun Ouyang
  12. Bradley C Naylor
  13. Joao A Paulo
  14. Katsuhiko Funai
  15. James E Cox
  16. Steven P Gygi
  17. Dennis R Winge
  18. Ralph J DeBerardinis
  19. Jared Rutter  Is a corresponding author
  1. Department of Biochemistry, United States
  2. Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center, United States
  3. Department of Cell Biology, Harvard University School of Medicine, United States
  4. Diabetes & Metabolism Research Center, United States
  5. Department of Nutrition and Integrative Physiology, United States
  6. Metabolomics, Proteomics and Mass Spectrometry Core Research Facilities University of Utah, United States
  7. Howard Hughes Medical Institute, United States
  8. Department of Internal Medicine, United States
Research Article
Cite this article as: eLife 2020;9:e58041 doi: 10.7554/eLife.58041
8 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
MtFAS is an essential pathway in mammalian skeletal myoblasts but does not contribute to synthesis of major cellular lipids.

(A) Schematic of the mitochondrial fatty acid synthesis pathway and downstream lipoic acid synthesis. (B) Crude isolated mitochondrial fractions from duplicate single cell clones of Mcat, Oxsm, and Mecr mutants, compared with GFP control clonal cell lines, were separated via SDS-PAGE and immunoblotted for the indicated targets. *=Lipoic acid band (reprobe of earlier blot). #=non specific bands C. GFP control and Oxsm mutant cells were infected with retroviral control plasmid (pCtrl) or a plasmid expressing Oxsm off the CMV promoter (pOxsm), plated at equal densities in normal growth medium with either 4.5 g/L glucose or 10 mM galactose and grown for 3 or 4 days, respectively, then stained with crystal violet. (D) Whole cell lysates from stable cell lines generated by infecting Oxsm mutant cells (OxsmΔ) or GFP controls with shRNA constructs targeting FASN (shFASN) or scramble control (shScramble) were separated by SDS-PAGE and immunoblotted for FASN, lipoylated proteins, or tubulin. (E-F) Stable cell lines created in (D) were incubated with U13C-glucose for the indicated number of doublings, harvested, lipids extracted, and analyzed via LC-MS. Shown are quantitation of m+2 isotopologues for two representative phospholipid species, PC 16:0_16:0 and PC 16:0_18:0. †=p < 0.001, ‡=p < 0.0001, error bars are SEM.

Figure 1—figure supplement 1
mtFAS is required for growth of cultured skeletal myoblasts but does not significantly contribute to cellular fatty acids.

(A) Quantification of T7E1 assay for editing efficiency in bulk transfected C2C12 cells with sgRNAs targeting the indicated mtFAS genes. (B) Quantification of single cell clone colony size 7 days after single cell sort. Small clones = <150 cells, Med = 150–500 cells, Large = >500 cells. (C) Cells were plated at 100,000 cells/plate in 10 cm dishes and counted every 24 hr. #=p < 0.01, all groups compared with control, †=p < 0.001, all groups compared with control, error bars SEM. (D-E) GFP control, Mcat, and Mecr mutant cells were infected with retroviral control plasmid (pCtrl) or plasmids expressing Mcat (D) or Mecr (E) off the truncated (Δ4,5CMV) or full CMV promoters as indicated. Cells were plated at equal densities in normal growth medium without glucose and with 10 mM galactose and grown for 4 days, then stained with crystal violet. (F) Sub-cellular fractionation of Mecr mutant cell lines and control. Whole cell lysate (WCL), post mitochondrial supernatant (PMS), and mitochondrial lysate (Mito) was isolated from each cell line indicated, normalized for total protein via BCA assay, and immunoblotted for Mecr or VDAC as a mitochondrial marker control. (G) Control (GFP) or Oxsm mutant (OxsmΔ) cells were stably infected with shRNA targeting FASN (shFASN) or control (shScramble) then incubated with U13C-glucose for one doubling were harvested, lipids extracted and saponified, and analyzed via FAMES analysis. Shown are quantitation of c16:0 and c18:0 fatty acids. ns = non significant, *=p < 0.05, #=p < 0.01, †=p < 0.001, error bars are SEM.

Figure 2 with 1 supplement
MtFAS mutants display profound loss of mitochondrial respiration and ETC complexes.

(A) Cells from three clones of each of the indicated genotypes were seeded in eight wells of a 96-well seahorse plate and allowed to adhere overnight, then equilibrated and treated with the indicated drugs following standard mitochondrial stress test protocols from the manufacturer to determine Oxygen Consumption Rate (OCR). #=p < 0.01, †=p < 0.001, ‡=p < 0.0001 all comparisons are to GFP control, error bars are SEM. (B) Cells of the indicated genotype were seeded in chambered coverglass slides, stained with Mitotracker Red and Mitotracker Green, and imaged. (C) Ratio of MitoTracker Red to MitoTracker Green fluorescence of cells from 30 fields of view quantified using Fiji ImageJ. *=p < 0.05, †=p < 0.001, ‡=p < 0.0001 all comparisons are to GFP control, error bars are SD. (D) Mitochondrial lysates generated from the indicated cell lines were normalized for total protein by BCA assay, incubated with 1% digitonin, then separated by blue-native PAGE and immunoblotted with the indicated antibodies.

Figure 2—figure supplement 1
ETC complex assembly and activity is regulated by interaction of LYRM proteins with ACP.

(A) Cells from three clones of each of the indicated genotypes were seeded in eight wells of a 96-well seahorse plate and allowed to adhere overnight, then equilibrated and treated with the indicated drugs following standard mitochondrial stress test protocols from the manufacturer to determine ExtraCellular Acidification Rate (ECAR). (B) Cells of the indicated genotype were seeded in chambered coverglass slides, stained with Mitotracker Red and Mitotracker Green, and imaged. Fluorescence of cells from 30 fields of view was quantified using Fiji ImageJ. *=p < 0.05, †=p < 0.001, ‡=p < 0.0001 all comparisons are to GFP control, error bars are SD. (C) Crude mitochondrial fractions were isolated from triplicate biological samples of mtFAS mutant and control cells using differential centrifugation. Isolated mitochondria were normalized for protein content via BCA assay and analyzed for Bc1-complex activity and citrate synthase activity (CS). ns = non significant, #=p < 0.01, †=p < 0.001, error bars are SD.

Figure 3 with 1 supplement
Posttranslational loss of ETC components in mtFAS mutants is specific to LYR proteins and their targets.

(A-B) Duplicate samples from the indicated cell lines were grown under proliferative conditions and subjected to TMT labeling and quantitative proteomics analysis. (A) Volcano plot of compiled Mecr clones vs. GFP controls showing all proteins (gray), mitochondrial proteins (blue), and electron transport chain subunits (ETC, red). Dashed gray lines indicate cutoffs for significance at -log10(p-value) = 1.3 and log2(Fold Change) = +/- 0.59. (B) Heatmap depicting log2(Fold Change) of OXPHOS subunits in the indicated cell lines. (C) Quadruplicate samples from mtFAS mutant cells and controls were grown under proliferative conditions. Total RNA was isolated, used as input for mRNA library prep, and sequenced. Resulting data were aligned to the mouse genome and analyzed for differential expression. ETC subunit-encoding transcripts are shown in red vs. all other transcripts (gray). (D-G) Relative abundance of the indicated LYR proteins or their targets in the indicated cell lines from the quantitative proteomics experiment described in (A-B) #=p < 0.01, †=p < 0.001, ‡=p < 0.0001, error bars are SD. All statistical comparisons shown are between mtFAS mutants and GFP-1 clone; p-values when compared with GFP-2 clone were similar or smaller than when compared with GFP-1 clone. (H) Crude mitochondrial lysates generated from the indicated cell lines by differential centrifugation were normalized for total protein by BCA assay, separated by SDS-PAGE, and immunoblotted with the indicated antibodies.

Figure 3—figure supplement 1
Transcription of ETC subunit encoding genes and translation of mitochondrially encoded proteins are unaffected in mtFAS mutants.

(A-C) Quadruplicate samples from mtFAS mutant cells and controls were grown under proliferative conditions. Total RNA was isolated, used as input for mRNA library prep, and sequenced. Resulting data were aligned to the mouse genome and analyzed for differential expression. ETC subunit encoding transcripts are shown in red vs. all other transcripts (black). (C) Cumulative distribution plots showing no difference in abundance between ETC encoding transcripts vs. all other genes for the indicated mtFAS mutant vs. GFP control, p>0.05, Wilcoxon rank-sum test. (D) HEK293T cells were transiently transfected with human NDUFAB1-Flag or empty vector control along with the indicated LYRM-V5 constructs, grown for 48 hr after transfection, harvested, and crude mitochondrial lysates were prepared by differential centrifugation. Flag immunoprecipitation was performed and eluates were blotted with the indicated antibodies alongside 10% of input. (E-H) Duplicate samples from the indicated cell lines were grown under proliferative conditions and subjected to TMT labeling and quantitative proteomics analysis. Relative abundance of the indicated LYR protein (E), LYR target (F), mitochondrially encoded proteins (G), and MALSU1 (H). *=p < 0.05, #=p < 0.01, error bars are SD. All statistical comparisons shown are between mtFAS mutants and GFP-1 clone.

Figure 4 with 1 supplement
Impairment of mtFAS promotes switch from oxidative to reductive mitochondrial metabolism.

(A–E) Triplicate biological samples from mtFAS mutant cell lines or GFP control were grown under standard proliferative conditions in high glucose medium (25 mM) and harvested for steady-state metabolomics analysis by LC-MS. Shown are relative pool sizes for the indicated metabolites. *=p < 0.05, #=p < 0.01, †=p < 0.001, ‡=p < 0.0001, error bars are SD. (F) Schematic of isotopomeric labeling of TCA cycle intermediates upon feeding with U13C-glutamine. Black circles indicate 13C carbons derived from labeled glutamine via oxidative TCA cycle flux (left). Purple circles indicate 13C carbons derived from labeled glutamine via reductive carboxylation (right). (G-L) Triplicate biological samples of the indicated genotype were labeled for 24 hr with U13C-glutamine, harvested, and analyzed via GC-MS for the indicated metabolites and their isotopologues. *=p < 0.05, #=p < 0.01, †=p < 0.001, ‡=p < 0.0001, error bars are SD.

Figure 4—figure supplement 1
Impairment of mtFAS causes changes in purine abundance and TCA cycle flux.

(A–E) Triplicate biological samples from mtFAS mutant cell lines or GFP control were grown under standard proliferative conditions in high glucose media (25 mM) and harvested for steady-state metabolomics analysis by LC-MS. Shown are relative pool sizes for the indicated metabolites. *=p < 0.05, #=p < 0.01, error bars are SD. (F-N) Triplicate biological samples of the indicated genotype were labeled with U13Cglutamine for 5 min, 15 min, 30 min, or 1 hr, harvested, and analyzed via GC-MS for the indicated metabolite isotopologues. *=p < 0.05, #=p < 0.01, †=p < 0.001, ‡=p < 0.0001, error bars are SD. Symbols indicate statistical comparison to GFP control in the order Mcat, Oxsm, Mecr.

Figure 5 with 1 supplement
MtFAS mutant phenotypes are not recapitulated by loss of lipoic acid alone.

(A) Immunoblot for the indicated proteins in whole cell lysates from Lipt1 mutant and control cell lines. (B) In triplicate experiments, cells of each of the indicated clones were seeded in eight wells of a 96-well seahorse plate and allowed to adhere overnight, then equilibrated and treated with the indicated drugs following standard mitochondrial stress test protocols from the manufacturer to determine Oxygen Consumption Rate (OCR). #=p < 0.01, †=p < 0.001, ‡=p < 0.0001 all comparisons are to GFP control, error bars are SEM. (C) Mitochondrial lysates generated from the indicated cell lines by differential centrifugation were normalized for total protein by BCA assay, incubated with 1% digitonin, then separated by BN-PAGE and immunoblotted with the indicated antibodies. (D) Four biological replicates from mtFAS mutant cell lines or GFP control were grown under standard proliferative conditions and harvested for steady-state metabolomics analysis by LC-MS. Shown are relative pool sizes for the indicated metabolites. *=p < 0.05, error bars are SD. (E) Four biological samples of the indicated genotype were labeled for 24 hr with U13Cglutamine, harvested, and analyzed via GC-MS for the indicated metabolites and their isotopologues. *=p < 0.05, #=p < 0.01, †=p < 0.001, error bars are SD.

Figure 5—figure supplement 1
Expression of mtFAS proteins is unchanged in Lipt1 mutant cell lines.

(A) Crude isolated mitochondrial proteins were isolated by SDS-PAGE and immunoblotted for MCAT, OXSM, and MECR, along with DNA polymerase gamma.

Figure 6 with 1 supplement
MtFAS is required for skeletal myoblast differentiation.

(A) Volcano plot of quantitative proteomics experiment showing Mecr mutant samples (n = 4) vs. GFP (n = 4) controls; all proteins (gray), and proteins associated with the differentiated skeletal muscle lineage (red). Dashed lines indicate cutoffs for significance at -log10(p-value) = 1.3 and log2(Fold Change) = +/- 0.59. (B-C) Cells of the indicated genotypes were plated and grown for 24 hr under proliferative conditions (day −1), then switched to 2% horse serum differentiation medium at 95% confluency (day 0). Medium was replaced daily for the indicated number of days. Plates were imaged (B) or whole cell lysates were collected at the indicated time points, separated by SDS-PAGE, and immunoblotted for the indicated proteins (C). (D-E) Quadruplicate samples from mtFAS mutant cells and controls were grown under proliferative conditions to 95% confluency then switched to 2% horse serum differentiation medium for 24 hr. Total RNA was isolated, used as input for mRNA library prep, and sequenced. Resulting data were aligned to the mouse genome and analyzed for differential expression. Transcripts for differentiation related proteins from panel (A) are shown in purple vs. all other transcripts (gray). (F) Relative steady state pool sizes of the indicated metabolites in mtFAS mutant cell lines vs. GFP controls grown in proliferative conditions from steady-state LC-MS metabolomics analysis. *=p < 0.05, #=p < 0.01, †=p < 0.001, ‡=p < 0.0001, error bars are SD.

Figure 6—figure supplement 1
Skeletal myoblast differentiation is delayed in mtFAS mutant cell lines.

(A) Cells of the indicated genotypes were plated and grown for 24 hr under proliferative conditions (day −1), then switched to 2% horse serum differentiation medium at 95% confluency (day 0). Medium was replaced daily for the indicated number of days. Plates were imaged at the indicated time points. (B-C) Quadruplicate samples from mtFAS mutant cells and controls were plated and allowed to adhere overnight, then harvested at Day −1 (proliferative,~70% confluency), Day 0 (~95% confluent), or Day one after switching to 2% horse serum differentiation medium for 24 hr. Total RNA was isolated, used as input for mRNA library prep, and sequenced. Resulting data were aligned to the mouse genome and analyzed for differential expression. Transcripts for differentiation related proteins from panel (A) are shown in purple vs. all other transcripts (gray). (B) Oxsm mutant vs. GFP controls after 24 hr in differentiating media. (C) Cumulative distribution plots of differentiation related transcripts vs. all other genes for the indicated genotypes, p>0.05, Wilcoxon rank-sum test. (D) Change in expression of transcripts from Day −1 to Day 0 for mtFAS mutant cell lines and GFP controls.

Author response image 1
Author response image 2

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
 cell line (M. musculus)C2C12ATCC#CRL-1772, RRID:CVCL_0188
 cell line (H. sapiens)HEK293TATCC#CRL-11268, RRID:CVCL_1926
 antibodyAnti-MCAT (mouse monoclonal)Santa Cruz#sc-390858, RRID:AB_2827536WB: (1:100)
 AntibodyAnti-OXSM (rabbit polyclonal)Thermo Fisher Scientific#PA5-32132, RRID:AB_2549605WB: (1:1000)
 AntibodyAnti-MECR (rabbit polyclonal)Proteintech#51027–2-AP, RRID:AB_615013WB: (1:1000)
 AntibodyAnti-Lipoic Acid (rabbit polyclonal)Abcam#ab58724, RRID:AB_880635WB: (1:1000)
 AntibodyAnti-DLAT (rabbit monoclonal)Abcam#ab172617, RRID:AB_2827534WB: (1:1000)
 antibodyAnti-NDUFAB1 (rabbit polyclonal)Abcam#ab96230, RRID:AB_10686984WB: (1:1000)
 AntibodyAnti-Flag (rabbit polyclonal)Sigma-Aldrich#F7425, RRID:AB_439687WB: (1:1000)
 AntibodyAnti-V5 (rabbit polyclonal)Abcam#ab9116, RRID:AB_307024WB: (1:2,000)
 AntibodyAnti-GRIM19 (mouse monoclonal)Abcam#ab110240, RRID:AB_10863178WB: (1:1,000)
 AntibodyAnti-SDHA (mouse monoclonal)Abcam#ab14715, RRID:AB_301433WB: (1:10,000)
 AntibodyAnti-UQCRFS1 (mouse monoclonal)Abcam#ab14746, RRID:AB_301445WB: (1:1000)
 AntibodyAnti-MTCO1 (mouse monoclonal)Abcam#ab14705, RRID:AB_2084810WB: (1:1000)
 AntibodyAnti-ATP5a (mouse monoclonal)Abcam#ab14748, RRID:AB_301447WB: (1:1000)
 AntibodyAnti-NDUFA9 (mouse monoclonal)Abcam#ab14713, RRID:AB_301431WB: (1:1000)
 AntibodyAnti-SDHB (mouse monoclonal)Abcam#ab14714, RRID:AB_301432WB:(1:2,000)
 AntibodyAnti-LYRM4
(rabbit polyclonal)
Thermo-Fisher#PA5-56448
RRID:AB_2643635
WB:(0.4 µg/mL)
 AntibodyAnti-VDAC (rabbit polyclonal)Cell Signaling#4866, RRID:AB_2272627WB: (1:1000)
 AntibodyAnti-CS (rabbit polyclonal)Abcam#ab96600, RRID:AB_10678258WB: (1:1000)
 AntibodyAnti-Lipoic Acid (rabbit polyclonal)Millipore#437695, RRID:AB_212120WB: (1:1000)
 AntibodyAnti-LIPT1 (rabbit polyclonal)Sigma-Aldrich#AV48784, RRID:AB_185290WB: (1:1000)
 AntibodyAnti-DLAT (mouse monoclonal)Cell Signaling#12362, RRID:AB_279789WB: (1:1000)
 AntibodyAnti-DLST (rabbit polyclonal)Cell Signaling#5556, RRID:AB_106951WB: (1:1000)
 AntibodyAnti-GAPDH (rabbit monoclonal)Cell Signaling#8884 RRID:AB_11129865WB: (1:2000)
 AntibodyAnti-MHC (mouse monoclonal)DSHB#SC-71, RRID:AB_2147165WB: (0.2 µg/ml)
 AntibodyAnti-Myogenin (mouse monoclonal)DSHB#F5D, RRID:AB_2146602WB: (0.5 µg/ml)
 AntibodyAnti-MyoD (mouse monoclonal)DSHB#D7F2, RRID:AB_1157912WB: (0.5 µg/ml)
 AntibodyGoat Anti-Mouse IgG (H and L) Antibody Dylight 800 ConjugatedRockland#610-145-002-0.5,
RRID:AB_10703265
WB:(1:10,000)
 AntibodyDonkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 680Invitrogen#A10043,
RRID:AB_2534018
WB:(1:10,000)
 AntibodyGoat Anti-Mouse IgG (H+L) Antibody, Alexa Fluor 680 ConjugatedInvitrogen#A21057, RRID:AB_141436WB:(1:10,000)
 AntibodyDonkey Anti-Rabbit IgG (H and L) Antibody Dylight 800 ConjugatedRockland#611-145-002-0.5, AB_11183542WB:(1:10,000)
 AntibodyGoat anti-Mouse IgG (H+L), Superclonal Recombinant Secondary Antibody, HRPThermo Fisher Scientific#A28177, RRID:AB_2536163WB:(1:10,000)
recombinant DNA reagentpMcat CRISPR sgRNA guide 1 (plasmid)This papersgRNA cloned in pLentiCRISPRv2GCGCGTCGCAATGAGCGCTC
recombinant DNA reagentpMcat CRISPR sgRNA guide 2 (plasmid)This papersgRNA cloned in pLentiCRISPRv2CGAGGCCGCGCACCGGGTAC
 recombinant DNA reagentpOxsm CRISPR sgRNA guide 1 (plasmid)This papersgRNA cloned in pLentiCRISPRv2CTAGTGACACCACTTGGCGT
 recombinant DNA reagentpOxsm CRISPR sgRNA guide 2 (plasmid)This papersgRNA cloned in pLentiCRISPRv2CGTTGGGACTCAACTAGTTT
 recombinant DNA reagentpMecr CRISPR sgRNA guide 1 (plasmid)This papersgRNA cloned in pLentiCRISPRv2AGGCTTGGTACCGCCACGGC
 recombinant DNA reagentpMecr CRISPR sgRNA guide 2 (plasmid)This papersgRNA cloned in pLentiCRISPRv2CGTGGCGGTACCAAGCCTCG
 recombinant DNA reagentpLipt1 CRISPR sgRNA guide 1 (plasmid)This papersgRNA cloned in pLentiCRISPRv2CACCGCTTCTAGATGTATGTGGTCG
 recombinant DNA reagentLipt1 CRISPR sgRNA guide 2 (plasmid)This papersgRNA cloned in pLentiCRISPRv2CACCGCTCCTTCTGTCGTCATCGGC
 recombinant DNA reagentpLentiCRISPRv2
(plasmid)
Addgene#52961 RRID:Addgene_52961
 recombinant DNA reagentpLKO.1 shFASN (plasmid)The Broad Institute#shRNA TRCN0000075703
 recombinant DNA reagentpshScramble (plasmid)Addgene#1864, RRID:Addgene_1864
 recombinant DNA reagentpsPAX2 (plasmid)Addgene#12259, RRID:Addgene_12259
 recombinant DNA reagentpDM2.G (plasmid)Addgene#12260, RRID:Addgene_12260
 recombinant DNA reagentpQXCIP mtDSRed (plasmid)This papermtDSRed in pQXCIP backbone
 recombinant DNA reagentpQXCIP-Δ4,5CMV-mMcat (plasmid)This paperMouse Mcat with truncated CMV promoter in pQXCIP backbone
 recombinant DNA reagentpQXCIP-CMV-mMcat (plasmid)This paperMouse Mcat with full CMV promoter in pQXCIP backbone
 recombinant DNA reagentpQXCIP-CMV-mOxsm (plasmid)This paperMouse Oxsm with full CMV promoter in pQXCIP backbone
 recombinant DNA reagentpQXCIP-Δ4,5CMV-mMecr (plasmid)This paperMouse Mecr with truncated CMV promoter in pQXCIP backbone
 recombinant DNA reagentpQXCIP-CMV-mMecr (plasmid)This paperMouse Mecr with full CMV promoter in pQXCIP backbone
 sequence-based reagentMcat CRISPR Ver FwdThis paperPCR primersGACCGACATGCAACTGCAAATAG
 sequence-based reagentMcat CRISPR Ver RevThis paperPCR primersGGCCAGTGAAGCCACAAAGA
 sequence-based reagentOxsm CRISPR Ver FwdThis paperPCR primersCAACCATGTTGTCAAAATGCTTG
 sequence-based reagentOxsm CRISPR Ver RevThis paperPCR primersGGTCTGAAACAGCAAAGCAGTTTC
 sequence-based reagentMecr CRISPR Ver FwdThis paperPCR primersGCTGTCGCGGACGAATG
 sequence-based reagentMecr CRISPR Ver RevThis paperPCR primersGTCGGAAGCATCCACTGAGAC
 commercial assay or kitTruSeq Stranded mRNA Library Prep kitIllumina#20020595
 commercial assay or kitTruSeq RNA UD IndexesIllumina#20022371
 commercial assay or kitNovaSeq S1 reagent KitIllumina#20027465
 commercial assay or kitKapa Library Quant KitKapa Biosystems#KK4824
 commercial assay or kitPierce BCA AssayThermo#23225
 commercial assay or kitD1000 ScreenTape assayAgilent Technologies5067–5583
 chemical compound, drug[U-13C]glutamineCambridge Isotopes#CLM-1822
 chemical compound, drug[U-13C]glucoseCambridge Isotopes#CLM-1396
 chemical compound, drugDigitoninGoldBio#D-180–2.5
 chemical compound, drugSPLASH LipidomixAvanti Polar Lipids#330707
 software, algorithmAgilent Mass Hunter Qual B.07.00Agilenthttps://www.agilent.com/en/products/software-informatics/masshunter-suite/masshunter/masshunter-software
 software, algorithmAgilent Mass Hunter Quant B.07.00Agilenthttps://www.agilent.com/en/products/software-informatics/masshunter-suite/masshunter/masshunter-software
 software, algorithmLipid AnnotatorAgilenthttps://www.agilent.com/en/products/software-informatics/mass-spectrometry-software/data-analysis/mass-profiler-professional-software
 software, algorithmMetaboAnalyst 4.0Xia and Wishart, 2016http://www.metaboanalyst.ca
 OtherLipofectamine 2000 transfection reagentInvitrogen11668019

Additional files

Supplementary file 1

Table of all steady-state metabolites measured in mtFAS mutants and controls.

All steady-state metabolites measured via LCMS. Values shown are average fold change from mean GFP abundance.

https://cdn.elifesciences.org/articles/58041/elife-58041-supp1-v2.docx
Supplementary file 2

Proteins associated with skeletal muscle differentiation are decreased in abudance in mtFAS mutants.

Relative expression of proteins associated with skeletal muscle differentiation from SL-TMT experiment. Values are arbitrary units.

https://cdn.elifesciences.org/articles/58041/elife-58041-supp2-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/58041/elife-58041-transrepform-v2.docx

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)