Volume 24, Issue 6, Pages 673-684.e4 (June 2017) The Mammalian Malonyl-CoA Synthetase ACSF3 Is Required for Mitochondrial Protein Malonylation and Metabolic Efficiency  Caitlyn E. Bowman, Susana Rodriguez, Ebru S. Selen Alpergin, Michelle G. Acoba, Liang Zhao, Thomas Hartung, Steven M. Claypool, Paul A. Watkins, Michael J. Wolfgang  Cell Chemical Biology  Volume 24, Issue 6, Pages 673-684.e4 (June 2017) DOI: 10.1016/j.chembiol.2017.04.009 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 673-684. e4DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

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 hr (mean ± SEM, n = 10). (D) Succinate secretion into the culture medium upon increasing dose of malonate for 24 hr (mean ± SEM, n = 4). Effect of concentration significant by two-way ANOVA. (E) [1,3-13C]Malonate flux. Percent abundance of m+1-labeled metabolites determined by liquid chromatography-tandem mass spectrometry that are significantly down-regulated in ACSF3 KO cells labeled with 2.5 mM [1,3-13C]malonate for 4 hr (mean ± SEM, n = 6). α-KG, α-ketoglutarate. n.s., not significant, *p < 0.05, **p < 0.001. Cell Chemical Biology 2017 24, 673-684.e4DOI: (10.1016/j.chembiol.2017.04.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

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 protein abundance in ACSF3 KO cells over control cells (mean ± SEM, n = 5, p < 0.05 are in bold). n.s., not significant, *p < 0.05, **p < 0.001. Cell Chemical Biology 2017 24, 673-684.e4DOI: (10.1016/j.chembiol.2017.04.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

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 hr (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 (5 mM) (mean ± SEM, n = 6, representative of two independent experiments, n.s., not significant, **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 (mean ± SEM, n = 12, pooled from two independent experiments, **p<0.01). (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 hr (mean ± SEM, n = 10, **p < 0.01 by posttest after two-way ANOVA). More metabolites and two-way ANOVA results in Table S1. Cell Chemical Biology 2017 24, 673-684.e4DOI: (10.1016/j.chembiol.2017.04.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 ACSF3 Is Required for Mitochondrial Protein Malonylation (A) [2-14C]Malonate incorporation into different classes of biomolecules by biphasic extraction after 4 hr 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 24 hr. 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. Cell Chemical Biology 2017 24, 673-684.e4DOI: (10.1016/j.chembiol.2017.04.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

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 24 hr before switching to malonate-free medium for the times indicated (0–24 hr 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 24 hr 5 mM malonate followed by incubation in malonate-free medium for the times indicated. Statistical significance for time and genotype effects determined by two-way ANOVA, n.s., not significant, **p < 0.01, ***p < 0.001. Cell Chemical Biology 2017 24, 673-684.e4DOI: (10.1016/j.chembiol.2017.04.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

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 24 hr. Total protein staining by amido black shown as loading control. (B and 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 24 hr. See also Figure S5. Cell Chemical Biology 2017 24, 673-684.e4DOI: (10.1016/j.chembiol.2017.04.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

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 (CV-Atp5a) and complex III component Uqcrc2 (CIII-Uqcrc2) shown as loading control. Representative of n = 3 experiments. Cell Chemical Biology 2017 24, 673-684.e4DOI: (10.1016/j.chembiol.2017.04.009) Copyright © 2017 Elsevier Ltd Terms and Conditions