The Mammalian Malonyl-CoA Synthetase ACSF3 Is Required for Mitochondrial Protein Malonylation and Metabolic Efficiency

Abstract

Malonyl-coenzyme A (malonyl-CoA) is a central metabolite in mammalian fatty acid biochemistry generated and utilized in the cytoplasm; however, little is known about noncanonical organelle-specific malonyl-CoA metabolism. Intramitochondrial malonyl-CoA is generated by a malonyl-CoA synthetase, ACSF3, which produces malonyl-CoA from malonate, an endogenous competitive inhibitor of succinate dehydrogenase. To determine the metabolic requirement for mitochondrial malonyl-CoA, ACSF3 knockout (KO) cells were generated by CRISPR/Cas-mediated genome editing. ACSF3 KO cells exhibited elevated malonate and impaired mitochondrial metabolism. Unbiased and targeted metabolomics analysis of KO and control cells in the presence or absence of exogenous malonate revealed metabolic changes dependent on either malonate or malonyl-CoA. While ACSF3 was required for the metabolism and therefore detoxification of malonate, ACSF3-derived malonyl-CoA was specifically required for lysine malonylation of mitochondrial proteins. Together, these data describe an essential role for ACSF3 in dictating the metabolic fate of mitochondrial malonate and malonyl-CoA in mammalian metabolism.

Keywords: ACSF3; CRISPR/Cas; SIRT5; acetyl-CoA; malonate; malonyl-CoA synthetase; malonylation; metabolomics; mitochondrial metabolism; succinylation.

Figure 1. ACSF3 is a Mitochondrial Malonyl-CoA Synthetase

(A) The reaction catalyzed by acyl-CoA synthetase family member 3 (ACSF3), a mammalian mitochondrial malonyl-CoA synthetase. (B) Immunoblot for ACSF3 in three control clones that were transfected with Cas9 only and three ACSF3 knockout (KO) clones. HSC70 is shown as a loading control. See Figure S1 for characterization of genomic mutations. (ns = non-specific band). (C) Steady-state intracellular malonate and succinate (D) concentrations, in the presence or absence of 5 mM malonate for 24 hours (mean ± SEM, n=10). (D) Succinate secretion into the culture medium upon increasing dose of malonate for 24h (mean ± SEM, n=4). Effect of concentration significant by 2-way ANOVA. (E) [1,3-¹³C]malonate flux. % abundance of m+1-labeled metabolites determined by LC-MS/MS that are significantly down-regulated in ACSF3 KO cells labeled with 2.5 mM 1,3-¹³C-malonate for 4 hours (mean ± SEM, n=6, α-KG = α-ketoglutarate). *p<0.05, **p<0.001

Figure 2. ACSF3 Regulates Mitochondrial Metabolic Efficiency

(A) Oxygen consumption rate of ACSF3 KO cells in the presence of 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate upon sequential administration of the specified mitochondrial inhibitors, normalized to cell number (mean ± SEM, n=5, representative of three independent experiments). (B) Oxidation of [U-¹⁴C]D-glucose, [U-¹⁴C]L-glutamine, and [U-¹⁴C]L-alanine to ¹⁴CO₂ in ACSF3 KO cells (mean ± SEM, n=6). (C) Immunoblot of several mitochondrial proteins. VDAC (voltage-dependent anion channel 1) as outer mitochondrial membrane marker; ATP5A component of ATP synthase, Complex III subunit 2 (UQCRC2), SDHB subunit of Complex II, and NDUFB8 subunit of Complex I of the inner mitochondrial membrane; aconitase (ACO2) and ACSF3 of the mitochondrial matrix; and HSC70 as cytoplasmic loading control. Protein abundance was normalized to HSC70, and values shown are fold change proteinabundance in ACSF3 KO cells over control cells (mean ± SEM, n=5, p<0.05 are in bold). *p<0.05, **p<0.001

Figure 3. Metabolic Alterations in ACSF3-Deficient Cells

(A) Acetyl-CoA carboxylase (ACC) phosphorylation (Ser79) in ACSF3 KO and control cells in the presence or absence of 5 mM malonate for 24 hours (mean ± SEM, n=6 from two independent experiments, **p<0.01 for genotypic effect, with no effect of malonate treatment, determined by posttest after two-way ANOVA). See also Figure S2. (B) [³H]acetate incorporation into total cellular lipids in the presence or absence of malonate (5mM) (mean ± SEM, n=6, representative of two independent experiments, **p<0.01 for genotypic effect, malonate treatment effect p=0.001 with significant interaction (p<0.05), determined by two-way ANOVA). (CPM = counts per minute). (C) [U-¹⁴C]glucose and [U-¹⁴C]glutamine incorporation into the total lipid fraction (mean ± SEM, n=6, *p<0.05). (D) [2-¹⁴C]malonate into total cellular lipids (n=12, pooled from two independent experiments). (E) Immunoblotting for lipoic acid-modified proteins, DLAT (E2 of pyruvate dehydrogenase) and DLST (E2 of α-ketoglutarate dehydrogenase), with HSC70 as loading control. (F) Select metabolite levels in the presence or absence of 5 mM malonate for 24 hours (mean ± SEM, n=10, **p<0.01 by posttest after two-way ANOVA). More metabolites and two-way ANOVA results in Table S1.

Figure 4. ACSF3 Is Required for Mitochondrial Protein Malonylation

(A) [2-¹⁴C]malonate incorporation into different classes of biomolecules by biphasic extraction after 4h incubation with 0.2 μCi at 0.1 mM malonate (mean ± SEM, n=3, representative of two independent experiments, *p<0.05, **p<0.01). See also Figure S2C. (B) Immunoblotting for malonylated lysine residues in whole cell and mitochondrial extracts of cells in the presence or absence of 5 mM malonate for 24h. ACSF3 KO cells were transiently transfected with a human ACSF3 expression vector, and control cells were transfected with a mitochondrially-targeted YFP. Total protein staining by amido black shown as loading control. See also Figure S3.

Figure 5. Mitochondrial Protein Malonylation Persists Following Malonate Washout

(A) Immunoblotting for malonylated lysine residues in mitochondrial extracts of cells treated with 5 mM malonate for 24h before switching to malonate-free media for the times indicated (0-24h after malonate washout). See also Figure S4. (B) Steady-state concentrations of cellular succinate, lactate, pyruvate, and threonine determined by ¹H-NMR (mean ± SEM, n=3) after 24h 5 mM malonate followed by incubation in malonate-free media for the times indicated. Statistical significance for time and genotype effects determined by two-way ANOVA, **p<0.01, ***p<0.001.

Figure 6. ACSF3 Does Not Affect Mitochondrial Protein Acetylation or Succinylation

(A) Immunoblotting for acetylated lysine residues in whole cell and mitochondrial extracts of cells in the presence or absence of 5 mM malonate for 24h. Total protein staining by amido black shown as loading control. (B-C) Immunoblotting for malonylated (B) and succinylated (C) lysine residues in mitochondrial extracts of ACSF3 KO and control cells treated with 5 mM malonate, 5 mM 3-nitropropanoate (3-NP, SDH suicide inhibitor), or both for 24h. See also Figure S5.

Figure 7. Tissue-specific Differences in Mitochondrial Protein Malonylation Reflect Acsf3 Abundance

(A) Immunoblot of malonylated lysine residues in mitochondrial extracts from liver, kidney, heart, and brown adipose tissue (BAT) of wild-type (WT) and ob/ob mice. Total protein staining by amido black shown as loading control. (B) Immunoblot of malonylated and succinylated lysine residues in mitochondrial extracts (mito) and total lysates (TL) of liver and BAT in WT and ob/ob mice. Complex V Atp5a subunit and Complex III component Uqcrc2 shown as loading control. Representative of n=3.