The one-carbon pool controls mitochondrial energy metabolism via complex I and iron-sulfur clusters

Abstract

Induction of the one-carbon cycle is an early hallmark of mitochondrial dysfunction and cancer metabolism. Vital intermediary steps are localized to mitochondria, but it remains unclear how one-carbon availability connects to mitochondrial function. Here, we show that the one-carbon metabolite and methyl group donor S-adenosylmethionine (SAM) is pivotal for energy metabolism. A gradual decline in mitochondrial SAM (mitoSAM) causes hierarchical defects in fly and mouse, comprising loss of mitoSAM-dependent metabolites and impaired assembly of the oxidative phosphorylation system. Complex I stability and iron-sulfur cluster biosynthesis are directly controlled by mitoSAM levels, while other protein targets are predominantly methylated outside of the organelle before import. The mitoSAM pool follows its cytosolic production, establishing mitochondria as responsive receivers of one-carbon units. Thus, we demonstrate that cellular methylation potential is required for energy metabolism, with direct relevance for pathophysiology, aging, and cancer.

Keywords: SLC25A26.

Fig. 1. Acute mitoSAM depletion is lethal through loss of mitoSAM-dependent metabolites.

(A) SAM import rates into larval mitochondria (n = 3) relative to the genetic background control wDah. (B) Four-day-old mutant Dm larvae. (C) Mitochondrial oxygen consumption rates in larvae (n = 5) normalized to protein content. (D) CoQ₉ levels in larval mitochondria (n = 3) normalized to protein content of input larvae. (E) Immunoblot on mitochondrial lysates from larvae, against lipoylation of pyruvate dehydrogenase (PDHc) and α-ketoglutarate dehydrogenase (αKGDHc) subunit E2, or porin as the loading control. (F) Total amino acid levels in p223l larvae relative to wDah controls (n = 3) or in serum of the previously reported p.V148G patient relative to mean standard values [diamonds; patient 1 in (9)]. Crossed shapes, metabolite not detected. CoA, coenzyme A. TCA, tricarboxylic acid. (G) Mitochondrial subsetting of larval RNA sequencing and proteomics data (n = 5). Expression level relative to wDah controls and vertical hierarchical clustering. UPRmt, mitochondrial unfolded protein response. Bar graphs show means + SD. *P < 0.05, **P < 0.01, and ***P < 0.001 with Dunnett’s test against wDah (A, C, and D). All experiments were performed on 4-day-old Dm larvae.

Fig. 2. SAMC is the only mitoSAM carrier and is required for OXPHOS and oxidative tricarboxylic acid (TCA) metabolism.

(A) Wild-type (Slc25a26^+/+) and Slc25a26 homozygous KO (Slc25A26^−/−, KO) embryos at embryonic day 8.5. (B) Blue Native polyacrylamide gel electrophoresis (BN-PAGE) immunoblot on MEF mitochondrial extracts with Coomassie staining (left) and an antibody mix against OXPHOS complex subunits (right) (n = 3). (C) Isolated respiratory chain complex activities (n = 3). (D) Bisulfite pyrosequencing on 12S mt-rRNA from MEFs (n = 3), or control (white) and Nsun4 KO (black) hearts (n = 1), targeting 4′-methylcytosine m⁴C909 (m⁴C) or 5′-methylcytosine m⁵C911 (m⁵C). (E) Western blot analysis on mitochondrial lysates from MEFs, showing lipoylation of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase E2 subunits. Hsp60 is the loading control. Short and long exposures are shown (n = 3). (F) Intracellular levels of α-ketoglutarate, citrate, and malate in MEFS cultured in ¹²C medium (n = 3). (G) Schematic of metabolite labeling obtained during reductive carboxylation of glutamine-glutamate–derived α-ketoglutarate. Circles represent ¹³C (black) and ¹²C (white) atoms. IDH, isocitrate dehydrogenase. (H) Mass isotopomers of α-ketoglutarate, citrate, and malate in MEFs cultured in ¹³C medium (n = 3). Bar graphs show means + SD. *P < 0.05 and ***P < 0.001 with two-sided Student’s t test of KO (black) against MEF control (white) cells. (C) and (D) show pooled data from three control and two KO MEF cell lines (n = 3).

Fig. 3. Cytosolic SAM production and not SAMC regulates the mitoSAM pool.

(A) mitoSAM levels upon RNAi knockdown or overexpression of cg4743 with corresponding transcript levels (two knockdown lines, one da-GAL4/+ control line, and two overexpression lines; n = 3). (B) Relative SAM import rates into enriched larval mitochondria upon uncoupling with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (n = 4). Control is 0 μM CCCP and was treated with dimethyl sulfoxide only. (C) Metabolic rescue of p223l larvae grown on standard yeast food supplemented with metabolites at the given concentration (n ≥ 10). Color indicates log₂-transformed fold change of larval size relative to controls, and circle size relates to observed larval size. Controls are larval size means of three independent vials. (D) Six-day-old p223l larvae grown on standard food with or without methionine supplementation. (E) Size quantification of larvae with or without methionine supplementation (n ≥ 10). A.U., arbitrary units. (F) Steady-state levels of SAM in total larval extracts and a mitochondrially enriched fraction with or without methionine supplementation (n = 3). (G) Cytosolic SAM levels (n = 4) in untreated larvae. Bar graphs show means + SD. n.s., not significant. *P < 0.05, **P < 0.01, and ***P < 0.001 with two-sided Student’s t test against wDah controls (E and F) or Dunnett’s test against control (A and B) or wDah (G). (A) to (C) and (E) to (G) performed on 4-day-old larvae. Met, 10 mM methionine.

Fig. 4. A methyl-SILAF map of mitochondrial protein methylation.

(A) Schematic overview of the workflow to detect the mitochondrial protein methylome. Mitochondrial or total protein extracts of 1:1 mixed unlabeled:labeled larvae were subjected to various protease digestions; in part high-pH reversed-phase liquid chromatography (HpH RPLC); antibody-based peptide enrichments against various l-lysine modifications (pmK) or monomethyl, symmetric, or asymmetric dimethyl l-arginine (mR, sdmR, and admR); liquid chromatography coupled to mass spectrometry; and bioinformatic data processing. (B) Number of modifications, sites, proteins, and their mitochondrial functional categories within the untargeted protein methylation library. (C) Relation of number of methylation events by number of protein members of the respective functional category. Blue dashed line shows the linear regression (R² = 0.72) with a 99% confidence interval in gray. Functional categories with an absolute standardized residual of more than one are labeled.

Fig. 5. The mitochondrial methylome is highly conserved from fly to mouse and human.

(A) Schematic workflow to confirm the untargeted mitochondrial fly methylome in three species by targeted PRM-based LC-MS/MS. Heavy isotope-labeled synthesized peptide standards were spiked into endogenous peptide samples. (B) Confidence scoring of selected methyl groups targeted with synthesized standards in Dm. (C) Conservation status of 203 methylated Dm residues in human protein homologs. nc, nonconserved amino acid. major ticks are 10, and minor ticks are 2 residues. (D) Scores of conserved residues in human (Hs) and mouse (Mm) fibroblasts as in (C). Listing in PhosphoSitePlus (68) as black squares in the literature column. The best score of at least three experiments is shown in (B) and (D). unmod, unmodified; mod, modified.

Fig. 6. ISC assembly is sensitive to mitoSAM loss.

(A) Interaction score of p223l transcriptomics and proteomics. Methylated proteins are shown in dark red. Gene names to methylated targets with an absolute interaction score of >1 in at least one dataset. (B) Mitochondrial aconitase activity (n = 4). (C) Transmission electron micrographs. Arrows indicate suspected iron deposits in null and p223l mitochondria. Scale bar, 0.5 μm. (D) Total cellular iron levels in 4-day-old Dm larvae (n = 4). (E) Interaction site of R72 in HsNFS1 with ISD11 and 4-PPE in gray [Protein Data Bank (PDB): 5USR]. (F) Cellular iron levels in fly models overexpressing DmNfs1 constructs (n = 3). Control is da-GAL4/+. (G) Immunoenrichment of DmNfs1.R77F-FLAG over DmNfs1.R77K-FLAG (n = 4). Black dots, mitochondrial; orange dot, DmNfs1. Significant proteins with known relation to iron metabolism or OXPHOS are labeled. (H) BN-PAGE immunoblot with an antibody against NFS1 in cells overexpressing HsNFS1-FLAG constructs (representative image, n = 3). The missing band in HsNFS1.R72K is marked. Graphs show means + SD. *P < 0.05, **P < 0.01, and ***P < 0.001 with Dunnett’s test against wDah (B and C) or control (F) or false discovery adjusted P value (G). (A) to (D), (F), and (G) performed on 4-day-old larvae.

Fig. 7. Reduced complex I methylation causes premature ageing.

(A) Codependency of SLC25A26 with genes of indicated categories in cancer cell lines as Pearson’s correlation of CERES scores summarized in box plots with median, upper and lower quartile, and 1.5× interquartile range as whiskers. (B) Modification status of the unmodified/modified endogenous peptides in SAMC KO MEFs versus controls relative to standards (n = 3). Significantly changed modified peptide in black. Targets in the right box without quantifiable unmodified peptides. (C) Proteomic levels of enzymes in serine biosynthesis and SAM metabolism in 4-day-old larvae (n = 5). (D) Fly survival curve, one of two experiments shown. (E) Adjusted P values of functional categories in female i172g fly proteomes at 30 or 50 days (n = 3). Significant labels (P < 0.05) are shown. (F) Proteomic changes in i172g insects [n = 5 for larvae (L), n = 3 for flies (F)] as violin plots with median as horizontal line. Graphs show means ±/+ SD. *P < 0.05 and **P < 0.01 with Bonferroni-adjusted P values (C and E) or log-rank test (D).