Fig. 4. Mitochondrial one-carbon metabolism is linked to functional ETC activity.
a Relative levels of intracellular serine and 5,10-methylenetetrahydrofolate (5,10-meTHF) in WT and ND1 mutant cells cultured either in glucose or galactose for 24 h and analyzed by LC-MS (n = 3). b Proteomics heatmap in WT cells exhibiting relative expression (log2 fold change) of proteins differentially regulated under 48 h galactose. c Measurement of formate production from serine using isolated mitochondria from WT, ND1, or SHMT2Δ cells (n = 4). d Cell number of WT, SHMT1Δ, and SHMT2Δ cell culture in galactose for 96 h (n = 3). e ME1 overexpression and 2 mM GSH supplementation rescues cell survival in galactose-grown SHMT2Δ cells (n = 3). f Seahorse analysis in WT sgNeg and SHMT2Δ in the absence or presence of 1 mM formate (n = 5). g GSH but not formate rescued cell survival in SHMT2Δ cells (n = 3). h GSH rescued cell number in ALDH1L2Δ cells cultured in galactose (n = 3). i ALDH1L2 converts 10-formyltetrahydrofolate to tetrahydrofolate and carbon dioxide in an NADP+-dependent reaction. ND1 cells display decreased serine-derived CO2 release compared to WT cells. ALDH1L2Δ cells were used as positive control (left panel). Galactose stimulated serine-derived CO2 release in WT cells but not in CI-deficient ND1 mutant cells (right panel) (n = 3). j Model illustrating the dependency of mitochondrial one-carbon metabolism on ETC function for NADPH production and how upregulation of ALDH1L2 stimulates NAPDH production in glucose-free conditions. Immunoblots shown are representative of >3 independent experiments, and all other experiments are represented as means ± SEM, n > 3 biological replicates. Asterisks denote *p < 0.05, **p < 0.01, or ***p < 0.001. Paired two-tailed Student’s t test in a and two-way ANOVA in c–j. Gluc glucose, Galac galactose, ser serine, Gly glycine, THF tetrahydrofolate, Pir Piericidin, Oligo oligomycin, Rot rotenone, Ant antimycin A. EV denotes empty vector. Red dashed lines indicate initial seeding density.