KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| ATF4 | Cell Signaling Technology | Cat#11815S; RRID: AB_2616025 |
| CHOP | Cell Signaling Technology | Cat##2895S; RRID: AB_2089254 |
| GAPDH | GeneTex | Cat# GTX627408; RRID: AB_11174761 |
| Calreticulin | Cell Signaling Technology | Cat#12238P; RRID: AB_2688013 |
| LAMP1 | Cell Signaling Technology | Cat#9091P; RRID: AB_2687579 |
| COXIV | Cell Signaling Technology | Cat#4850S; RRID: AB_2085424 |
| Beta Tubulin | GeneTex | Cat# GTX101279; RRID: AB_1952434 |
| Beta Actin | GeneTex | Cat#GTX109639; RRID: AB_1949572 |
| Vinculin | Cell Signaling Technology | Cat#4650S; RRID: AB_10559207 |
| CHKA | Cell Signaling Technology | Cat#13422S; RRID: AB_2798216 |
| CDIPT | GeneTex | Cat#GTX80599; RRID: AB_11166614 |
| PCYT1A | Sigma-Aldrich | Cat#HPA035428; RRID: AB_10602045 |
| HMOX1 | Proteintech | Cat#10701–1-AP; RRID: AB_2118685 |
| FTH1 | Cell Signaling Technology | Cat#4393S; RRID: AB_11217441 |
| FLVCR1 | Santa Cruz Biotechnology |
Cat#sc-390100 |
| NDUFS1 | Proteintech | Cat#12444–1-AP; RRID: AB_2282657 |
| OPA1 | BD Biosciences | Cat#612606; RRID: AB_399888 |
| MRPS35 | Proteintech | Cat#16457–1-AP; RRID: AB_2146521 |
| SLC44A1 | ABclonal | Cat#A15413; RRID: AB_2762321 |
| CHT1/SLC5A7 | Santa Cruz Biotechnology |
Cat#33713; RRID: AB_2189835 |
| Citrate Synthase | Cell Signaling Technology | Cat#14309; RRID: AB_2665545 |
| FLAG | Sigma-Aldrich | Cat#F1804; RRID: AB_262044 |
| Anti-Mouse IgG-HRP | Cell Signaling Technology | Cat#7076; RRID: AB_330924 |
| Anti-Rabbit IgG-HRP | Cell Signaling Technology | Cat#7074; RRID: AB_2099233 |
| AlexaFluor 568 Anti-Mouse | Invitrogen | Cat#A10037; RRID: AB_2534013 |
| AlexaFluor 488 Anti-Rabbit | Invitrogen | Cat#A21206; AB_2535792 |
| Bacterial and virus strains | ||
| NEB Stable Competent E. coli | New England Biolabs | Cat#C3040 |
| Biological samples | ||
| Chemicals, peptides, and recombinant proteins | ||
| RPMI 1640 Culture Media | Gibco | Cat#11875–093 |
| Fetal Bovine Serum | Sigma-Aldrich | Cat#12306C |
| Penicillin Streptomycin | Gibco | Cat#15140–122 |
| Trypsin | Gibco | Cat#25200 |
| RPMI 1640 Culture Medium w/o L-Glutamine, L-Methionine, Choline Chloride, Folic Acid and Vitamin B12 |
US Biological Life Sciences | Cat#R8999–21 |
| L-glutamine | Alfa Aesar | Cat#J60573 |
| L-methionine | Alfa Aesar | Cat#J61904 |
| Choline Chloride | MP Biomedicals | Cat#101386 |
| Folic Acid | Alfa Aesar | Cat#J62937 |
| Vitamin B12 | Alfa Aesar | Cat#A14894 |
| Dialyzed Fetal Bovine Serum | Gibco | Cat#26400–044 |
| Phosphocholine chloride calcium salt tetrahydrate | Sigma-Aldrich | Cat#P0378 |
| RSM932A | Cayman Chemical | Cat#215518 |
| Tunicamycin | Tocris | Cat#3516 |
| Oligomycin | Millipore | Cat#495455 |
| Hemin | Sigma-Aldrich | Cat#H9039 |
| FCCP | BioVision | Cat#2398 |
| Succinylacetone | Cayman Chemical | Cat#24401 |
| Hemicholinium-3 | Sigma-Aldrich | Cat#H108 |
| Zinc (II) Mesophoryrin IX (ZnMP) | Frontier Scientific | Cat#M40628 |
| Ammonium Iron (III) Citrate (FAC) | Sigma-Aldrich | Cat#F5879 |
| BsmBI-v2 | New England Biolabs | Cat#R0739L |
| BamHI-HF | New England Biolabs | Cat#R3136L |
| NotI-HF | New England Biolabs | Cat#3189L |
| Gibson Assembly Master Mix | New England Biolabs | Cat#E2611L |
| T4 Ligase | New England Biolabs | Cat#M0202 |
| X-tremeGENE 9 DNA Transfection Reagent | Roche | Cat#6364787001 |
| Polybrene | Sigma-Aldrich | Cat#H9268 |
| Puromycin | Sigma-Aldrich | Cat#P8833 |
| Blasticidin | Invivogen | Cat#ant-bl-1 |
| Protease Inhibitor | EMD Millipore | Cat#535140 |
| Protease Inhibitor | Sigma-Aldrich | Cat#11836170001 |
| Phosphatase Inhibitor | Roche | Cat#490685001 |
| Poly-D-Lysine | ChemCruz | Cat#sc-136156 |
| LipidTOX Deep Red | Invitrogen | Cat#H34477 |
| Prolong Gold Antifade Mountant | Invitrogen | Cat#P10144 |
| ECL Chemiluminescence | Perkin Elmer | Cat#NEL105001EA |
| ECL Chemiluminescence | Cytiva | Cat#RPN2232 |
| Donkey Serum | Jackson ImmunoResearch |
Cat##017–000-12 |
| Tetramethylrhodamine, methyl ester, perchlorate (TMRM) | Invitrogen | Cat#T668 |
| [1,2-13C2]Choline Chloride | Cambridge Isotope Laboratories | Cat#CLM-548.01 |
| Heavy Labeled Amino Acid Internal Standards | Cambridge Isotope Laboratories | Cat#MSK-A2–1.2 |
| HPLC Grade Water | Fisher Scientific | Cat#W6 |
| HPLC Grade Methanol | Fisher Scientific | Cat#A454–1 |
| HPLC Grade Acetonitrile | Fisher Scientific | Cat#A955–1 |
| [Methyl-3H]-Choline Chloride | Perkin Elmer | Cat#NET109001MC |
| Insta-Gel Plus Scintillation Cocktail | Perkin Elmer | Cat#601339 |
| Krebs-Ringer Buffer | Alfa Aesar | Cat#J67795 |
| Pierce Anti-HA Magnetic Beads | Thermo Scientific | Cat#88837 |
| Modified Davidson’s Fixative | Electron Microscopy Sciences | Cat#64133–50 |
| Critical commercial assays | ||
| Pierce BCA Protein Assay Kit | Thermo Fisher Scientific |
Cat#23227 |
| Choline Kinase Assay Kit | Abcam | Cat#ab239729 |
| RNeasy Mini Kit | Qiagen | Cat#74104 |
| TruSeq Stranded mRNA Library Prep | Illumina | Cat#20020595 |
| CellTiter-Glo Luminescent Cell Viability Assay | Promega | Cat#G7571 |
| DNeasy Blood & Tissue Kit | Qiagen | Cat#69506 |
| Deposited data | ||
| RNAseq | GEO | Accession #:GSE228126 |
| MitoIP TMT Proteomics | PRIDE | Accession #:1–20230329-151157 |
| Uncropped western blots and raw values used to generate all graphs | This Paper | Data S1 – Source Data |
| Experimental models: Cell lines | ||
| HeLa | ATCC | CCL-2 |
| HEK293T | ATCC | CRL-1573 |
| Experimental models: Organisms/strains | ||
| Mouse: C57BL/6JSmoc-Flvcr1em1Smoc | Dr. Janis Abkowitz (University of Washington)9 |
N/A |
| Oligonucleotides | ||
| sgRNA, cDNA, and Genotyping Oligonucleotides | This Study | See Table S1 |
| Recombinant DNA | ||
| plentiCRISPR-v2 puro | Addgene | Cat#982990 |
| plentiCRISPR-v1 GFP | Addgene | Cat#75159 |
| pMXS-IRES-Blast | Cell Biolabs | Cat#RTV-016 |
| pMXS-3xHA-OMP25-mCherry | 42 | N/A |
| pMXS-3xMyc-OMP25-mCherry | 42 | |
| pLV-EF1a-RFP | Dr. Navdeep Chandel (Northwestern University)46 |
N/A |
| pLV-EF1a-RFP-AOX | Dr. Navdeep Chandel (Northwestern University)46 |
N/A |
| plentiCRISPR-v1 GFP FLVCR1_sg5 | This Study | N/A |
| plentiCRISPR-v2 Puro FLVCR1_sg5 | This Study | N/A |
| plentiCRISPR-v2 Puro CHKA_sg2 | This Study | N/A |
| plentiCRISPR-v2 Puro KIAA0141/DELE1_sg1 | This Study | N/A |
| plentiCRISPR-v2 Puro KIAA0141/DELE1_sg2 | This Study | N/A |
| plentiCRISPR-v2 Puro EIF2AK3/PERK_sgRNA | This Study | N/A |
| plentiCRISPR-v2 Puro EIF2AK2/PKR_sgRNA | This Study | N/A |
| plentiCRISPR-v2 Puro GCN2_sgRNA | This Study | N/A |
| plentiCRISPR-v2 Puro EIF2AK1/HRI_sgRNA | This Study | N/A |
| plentiCRISPR-v2 Puro SPNS1_sg1 | This Study | N/A |
| plentiCRISPR-v2 Puro SPNS1_sg2 | This Study | N/A |
| pMXS-IRES-Blast FLVCR1/FLVCR1a | This Study | N/A |
| pMXS-IRES-Blast FLVCR1b | This Study | N/A |
| pMXS-IRES-Blast FLVCR2 | This Study | N/A |
| pMXS-IRES-Blast SLC44A1 | This Study | N/A |
| pMXS-IRES-Blast SLC5A7 | This Study | N/A |
| pMXS-IRES-Blast CHKA | This Study | N/A |
| pMXS-IRES-Blast CG1358 | This Study | N/A |
| Human CRISPR Metabolic Gene Knockout Library | Addgene | Cat#110066 |
| Software and algorithms | ||
| FCS Express 7 | DeNovo Software |
https://denovosoftware.com/; RRID: SCR_016431 |
| Skyline Daily v22 | MacCoss Lab |
https://skyline.ms/project/home/software/Skyline/begin.view; RRID: SCR_014080 |
| Prism 8 | GraphPad |
https://www.graphpad.com/scientificsoftware/prism; RRID: SCR_002798 |
| XCaliburQual Browser 2.2 | Thermo Scientific | https://www.thermofisher.com/order/catalog/product/OPTON-30965#/OPTON-30965; RRID: SCR_014593 |
| LipidSearch 4.2 | Thermo Fisher Scientific / Mitsui Knowledge Industries |
https://www.thermofisher.com/order/catalog/product/OPTON-30879?SID=srchsrp-OPTON-30879; |
| Proteome Discoverer v 2.5 | Thermo Fisher Scientific | https://www.thermofisher.com/order/catalog/product/OPTON-31041?SID=srch-hj-OPTON-31041 |
| Other | ||
| Z2 Coulter Counter | Beckman | Model Z2 |
| SpectraMax Microplate Reader | Molecular Devices | Model M3 |
| Primovert Microscope | Carl Zeiss | 415510-1105-000 |
| Multiphoton Microscope with Confocal Modality | Nikon | A1R MP |
| Tecnai G2-12 Transmission Electron Microscope with AMT BioSprint29 Digital Camera | FEI | G2-12 |
| FACSAriaII | BD Biosciences | 643181 |
| Attune NxT Flow Cytometer | Invitrogen | N/A |
| Bead Ruptor 24 | Omni International | N/A |
| Dionex UltiMate 3000 UPLC System | Thermo Fisher Scientific | N/A |
| Tri-Carb 2910 TR Scintillation Counter | Perkin Elmer | N/A |
| PicoLab Rodent Diet | PicoLab | Cat#5053; See Data S1 for composition |
| Choline Supplemented (20g/kg) Iron-Supplemented L-AA defined Rodent Diet | Dyets | Cat#519586; See Data S1 for composition |
| Choline Sufficient (14.48g/kg) Iron-Supplemented L-AA defined Rodent Diet | Dyets | Cat#518754; See Data S1 for composition |