METHOD DETAILS

To screen for genetic suppressors of gas-1(fc21) in hyperoxia thousands of L4 animals were exposed to 47 mM EMS (ethyl methanesulfonate) (Sigma M0880) for four hours while rocking. Animals were then washed twice with M9 buffer and allowed to recover on standard NGM plates. F1 animals were bleach prepped as described above to generate a synchronized L1 stage population of mutagenized F2 animals, which were then dropped onto standard NGM plates at 50% oxygen. Plates were checked daily and F2 individuals capable of growing to adulthood were transferred onto new plates. Fertile isolates were retested using F3 or F4 progeny to confirm their phenotype and then genomic DNA for whole genome sequencing was isolated using Gentra Puregene Tissue Kit (Qiagen 158667).

To identify candidate suppressor mutations in screen isolates we sheared genomic DNA using a Covaris S2 sonicator and prepared libraries using the NEBNext DNA library prep kit for Illumina. Libraries with unique barcodes were quantified using the Qubit dsDNA HS Assay Kit (Life Technologies {"type":"entrez-protein","attrs":{"text":"Q32851","term_id":"75280859","term_text":"Q32851"}}Q32851) and pooled in sets of 24 and sequenced using Illumina HiSeq⁴⁸. Raw FASTQ files were analyzed on the Galaxy platform (usegalaxy.org) with the following workflow: TrimGalore! to trim reads, Map with BWA to align reads to the C. elegans reference genome, MiModD to call variants, and SnpEff to identify mutations that may affect protein function. Lists of protein-altering mutations from each suppressor strain were then compared to identify genes with multiple mutant alleles. These candidate genes were then verified using targeted CRISPR/Cas9-based editing.

For protein sequence comparisons, homologs were identified using BlastP and, in the case of NDUFS2, from https://caenorhabditis.org/⁴⁹ and alignments made with ClustalW. For annotation of C. elegans CMTR-1 and human CMTR1 sequence features, predicted nuclear localization signals were identified using cNLS mapper⁵⁰ and intrinsically disordered domains were identified using both DEPICTER⁵¹ and PrDOS⁵².

To measure C. elegans growth and development crowded plates of gravid animals were washed into tubes in M9 buffer [3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 ml 1 M MgSO₄, H₂O to 1 liter] and incubated with 20% bleach and 10% 5M KOH for 5 minutes while vortexing. The resulting embryos were washed 3x in M9 buffer and allowed to hatch overnight while rocking in M9. The following day arrested L1 animals were dropped onto E. coli OP50 plates and incubated at 20°C. For assays in hyperoxia (50% or 100% oxygen) plates were sealed in a modular chamber (Stemcell Technologies #27310) and flushed for 3 minutes with either a 50:50 mixture of oxygen and nitrogen, or with pure oxygen gas. For assays in hypoxia (1% oxygen), animals were incubated in a Hypoxic in vitro cabinet (Coy Laboratory Products, Inc) at room temperature. To measure animal length, images were acquired using a ZEISS Axio Zoom V16 microscope with ZEN PRO software and the midline of individual animals was quantified in FIJI software. Brood size measurements were made by transferring adult C. elegans to new plates daily and counting all progeny generated over the course of egg-laying adulthood.

To measure hsp-6::gfp and nduf-2.2::gfp fluorescence, animals were mounted on agar pads, immobilized in sodium azide, and imaged at 70x magnification using a ZEISS Axio Zoom V16 microscope with ZEN PRO software. Fluorescent images were quantified by calculating the mean fluorescence along the midline of the intestine (for hsp-6::gfp) or by calculating the maximum fluorescence in embryos (for nduf-2.2::gfp) using FIJI software. To visualize cmtr-1::gfp localization patterns, images were acquired with a Nikon A1R confocal microscope, using a 60X/1.49 NA oil objective using 488nm excitation at 290nm/pixel. Worms were immobilized on 10% agarose pads with 0.3 μl of 0.1 μm diameter polystyrene microspheres (Polysciences 00876-15, 2.5% w/v suspension). To quantify colocalization of cmtr-1(ΔG-patch)::gfp and dcap-1::DsRed, green or red foci from three independent animals were blindly selected and the presence or absence of foci in the other channel was scored.

To measure nduf-2.2 mRNA levels by qPCR, total RNA was isolated from mixed stage animals using TRIzol Reagent (ThermoFisher 15596026). RNA was DNase treated using DNA-free Kit (Invitrogen AM1906) and cDNA was synthesized by the ProtoScript II First Strand cDNA Synthesis Kit (NEB E6560). Quantitative real-time PCR was performed using iQ SYBR Green Supermix (Biorad) on a BIORAD CFX Real-Time System. Three biological replicates were analyzed for each genotype, and three technical replicates per sample were performed in each qPCR run. Negative controls included (1) cDNA samples synthesized without reverse transcriptase and (2) cDNA synthesized from nduf-2.2 deletion mutants, confirming that the nduf-2.2 primers were specific. Delta Cq values were normalized to the housekeeping gene rps-23. All primer sequences are available in the Key Resources Table.