Fig 2: SLC25A39 is required for mitochondrial glutathione transport.Volcano plots of mitochondrial metabolites from the A39 KO and control cells in K562 cells (a) and HeLa cells (b) highlighting a depletion of reduced and oxidized glutathione, GSH and GSSG, respectively, in the A39 KO mitochondria. c schematic of the biosynthesis and degradation of glutathione in mammalian cells. GSH is a tripeptide ?-Glu-Cys-Gly exclusively synthesized in the cytosol by two cytosolic enzyme systems, ?-glutamylcysteine synthetase (GCLC and GCLM) and GSH synthetase (GSS) d LC-MS measurement of reduced GSH, oxidized GSSG and GSH/GSSG ratio in isolated mitochondria (n = 3) and whole cells (n = 4). e Schematic overview of the organelle-based GSH uptake assay. Note that the imported labeled GSH can react with endogenous, unlabeled GSH to generate GSSG. f LC-MS measurement of mitochondrial labeled GSH and singly labeled GSSG in the uptake assay (30 min) (n = 3). g Time-course of labeled GSH uptake into mitochondria (n = 3). h GSSG isotopomer distribution from the inside (washed mitochondria fraction) and the outside (assay supernatant) of the control mitochondria in the GSH uptake assay at the 30 min time point (n = 3). Statistical significance was calculated using two-tailed t test. Significance level were indicated as *** p < 0.001, ** p < 0.01, * p < 0.05 and n.s. p > 0.05. Data are expressed as mean ± SD. Source data are provided as a Source Data file.

Fig 3: Identification of Gene x Gene x Environment (GxGxE) interactions.a Heatmap shows the p-score for pairwise genetic interaction. The values are z-transformed to compare across media conditions, as described in the methods. For comparison, a positive control pair (BCL2L1 x MCL1) that are always detrimental when jointly knocked out and another pair (UCP1 x SLC25A31) that do not interact are shown. The rows and columns are not clustered. b LFC values from the CRISPR screen are shown to illustrate the strongest synthetic sick genetic interaction observed from the CRISPR screen, SLC25A5 and SLC25A6 loss in the galactose condition. The red line indicates the expected LFC, based on an additive model, if there were no genetic interaction. The black line indicates the actual median LFC value. c Follow-up validation of the genetic interaction between SLC25A5 and SLC25A6 in the galactose (pctrl/A5KO = 0.59, pctrl/A6KO = 0.66, pA5KO/DKO = 2.11 × 10-7, pA6KO/DKO = 1.93 × 10-8). The dotted line indicates the expected growth fitness, based on an additive model, if there were no specific genetic interaction (n = 3). d The cartoon illustrates the function of ANTs in supporting bioenergetics. e Transcript levels of the four ANTs in K562 cells (data plotted from the published PBS vehicle treated K562 cells RNA-seq dataset GSE74999) (n = 3). f qPCR of SLC25A4 mRNA level in SLC25A5 KO and SLC25A5 and SLC25A6 DKO cells (n = 3). g Follow-up validation of the genetic interaction between SLC25A37 and SLC25A39 in the glucose (pctrl/A37KO = 1.04×10-5, pctrl/A39KO = 3.08×10-6, pA37KO/DKO = 0.56, pA39KO/DKO = 0.0092) and antimycin (pctrl/A37KO = 0.00074, pctrl/A39KO = 0.0498, pA37KO/DKO = 5.54×10-5, pA39KO/DKO = 0.030) conditions (n = 6). Statistical significance was calculated using two-tailed t test. Significance level were indicated as *** p < 0.001, ** p < 0.01, * p < 0.05 and n.s. p > 0.05. Data are expressed as mean ± SD. Source data are provided as a Source Data file.

Fig 4: Structural modeling and functional mutagenesis of SLC25A39.a Multiple sequence alignment of the eukaryotic A39 ortholog sequences spanning diverse taxa alongside with the human amino acid ornithine transporter SLC25A15 (ORNT1) and the human citrate transporter SLC25A1 (TXTP). The positions for the human SLC25A39 Arg225 and Asp226 residues predicted to bind amino acids are labeled by asterisks. b The modeled human A39 structure based on the ANT structure c-state conformation (PDB: 1OKC), in both side view (left) and cytoplasmic/mitochondrial intermembrane space (IMS) view (right). Odd transmembrane domain (TM1, 3, 5) are shown in gray and even (TM2, 4, 6) are shown in cyan. Two predicted GSH binding residues (R225, D226) and the predicted residue critical for solute-binding induced conformational change (K329) are shown. The predicted interaction between R225 and D226 with the carboxylate and amino group of the glutamate residue of GSH is shown by dashed line. c Western blotting showing the endogenous A39 (#) and ectopically expressed A39 protein (*). VDAC was used as the loading control. d LC-MS measurement of mitochondrial GSH level showing that the predicted substrate binding mutant A39D226A cannot restore mitochondrial GSH level in the A39 KO cells (n = 3). Statistical significance was calculated using two-tailed t test. Significance level were indicated as *** p < 0.001, ** p < 0.01, * p < 0.05 and n.s. p > 0.05. Data are expressed as mean ± SD. Source data are provided as a Source Data file.