Fig 1: The subcellular distribution of metabolic enzymes and their putative interactors reveals their nuclear neighborhood.(A and B) Putative interactor signal (biotinylation signal measured using fluorescently labeled streptavidin) in the nuclear region of whole cells (A) or isolated nuclei (B) expressing the bait enzymes. “ni” refers to cells where the expression of the bait enzymes was not induced. (C) Representative immunofluorescence images and quantification of ACO2, OGDH, and IDH3G in the nucleus of mouse embryonic stem cells. The quantification was based on immunofluorescence using antibodies against the endogenous enzymes. “2ary” refers to cells stained only with the secondary, fluorophore-conjugated, antibody. DNA was stained with Hoechst (cyan). Scale bars, 15 µm. (D and E) Representative immunofluorescence images and quantification of OGDH (D) and succinylation (E) in naïve mouse embryonic stem cells and in differentiated counterparts. Scale bars, 10 µm. In (A) to (E), points represent quantification (log2 scale) of individual cells or nuclei, with the population mean and SD calculated for each case. In (B), (D), and (E), the biotinylation, OGDH, and succinylation levels were normalized to Hoechst signal. In (A) to (E), significance was assessed using the Wilcoxon rank sum test. a.u., arbitrary units.
Fig 2: The nTCA cycle is incomplete and different from the mitochondrial TCA cycle. a HepG2 cells were immunostained with antibodies against CS, ACO2, IDH3A, OGDH, SUCLG2, FH, or MDH2. The mean immunofluorescent intensity of the indicated protein in mitochondria and nucleus was analyzed by Image J software. Error bars represent mean ± SD for triplicate experiments. b Nuclei isolated from ACO2-deficient HepG2 cells were incubated with 13C6-citrate and acetyl-CoA for detection of 13C-labeled metabolic intermediates related to the TCA cycle by LC-MS. The data were normalized to protein concentration. Error bars represent mean ± SD for triplicate experiments (*p < 0.05). Western blot analysis was utilized to determine the expression of indicated protein. c Nuclei isolated from MDH2-deficient HepG2 cells were incubated with 13C4-malate and acetyl-CoA for detection of 13C-labeled metabolic intermediates related to the TCA cycle by LC-MS. The data were normalized to protein concentration. Error bars represent mean ± SD for triplicate experiments (*p < 0.05). Western blot analysis was utilized to determine the expression of indicated protein. d Mitochondria and nuclei isolated from an equal number of HepG2 cells were incubated with oxaloacetate and acetyl-CoA for measurement of ATP production using the ATP Colorimetric/Fluorometric Assay Kit. Error bars represent mean ± SD for triplicate experiments (*p < 0.05)
Fig 3: The nuclear TCA cycle-associated enzymes catalyze their corresponding biochemical reactions. a HepG2 cells treated with siRNA against control, CS, ACO2, IDH2, IDH3A, OGDH, SCS, SDHA, FH, MDH2 or GDH, and the nuclear and cytoplasmic extracts were prepared from HepG2 cells for enzymatic activity assays using the corresponding kit. Error bars represent mean ± SD for triplicate experiments (*p < 0.05). b The metabolic intermediates related to the TCA cycle were detected in HepG2 nuclei by LC-MS. c HepG2 nuclei were incubated with oxaloacetate and 13C3-pyruvate, 13C6-citrate, or 13C4-fumarate for detection of 13C-labeled-metabolic intermediates related to the TCA cycle by LC-MS. The data were normalized to protein concentration. Error bars represent mean ± SD for triplicate experiments (*p < 0.05). d HepG2 nuclei were incubated with or without oxaloacetate (OAA) and acetyl-CoA for detection of citrate and isocitrate by LC-MS. The data were normalized to protein concentration. Error bars represent mean ± SD for triplicate experiments (*p < 0.05). e Nuclei isolated from CS-depleted HepG2 cells were incubated with or without oxaloacetate and acetyl-CoA for detection of citrate and isocitrate by LC-MS. The data were normalized to protein concentration. Error bars represent mean ± SD for triplicate experiments (*p < 0.05). The efficiency of CS siRNA was determined by western blotting analysis. f Nuclei isolated from CS-depleted HepG2 cells were incubated with or without oxaloacetate and 13C3-pyruvate for detection of 13C-labeled-citrate by LC-MS. The data were normalized to protein concentration. Error bars represent mean ± SD for triplicate experiments. g Affinity-purified CS from HepG2 nuclei was incubated with 5 mM oxaloacetate and 0–40 µM acetyl-CoA, and the production of citrate was measured using the Citrate Colorimetric/Fluorometric Assay Kit. Error bars represent mean ± SD for triplicate experiments
Fig 4: Proximity biotinylation MS reveals core nuclear proteins as putative interactors of IDH1, IDH3G, OGDH, and ACO2.(A) Overview of the identified interactors of each bait enzyme. Heatmap shows fold changes in the abundance of each putative interactor in cells expressing the bait enzyme compared to the parental cells. Top: Significantly enriched GO terms for cellular component (top) and “biological process” (bottom) are shown for each cluster. (B) Subcellular distribution of the putative interactors of each bait enzyme. Nucleus and mitochondria refer to proteins detected only in the nucleus and mitochondria, respectively. Nucleus shared, proteins detected in the nucleus and any other compartment. Mitochondria shared, proteins detected in the mitochondria and any other compartment excluding nucleus. (C) Correlation between the percentage of interacting nuclear proteins and the abundance of the bait enzymes; the latter was estimated using immunofluorescence (main panel) or proteomics (inset). (D) Correlation between the percentage of interacting nuclear proteins and the biotinylation levels in cells expressing the bait enzymes. (E) Selected putative interactors of the bait enzymes. (F) Subcellular distribution of succinylated peptides in U2OS cells quantified by LOPIT. Inset: Enriched GO terms for nuclear proteins with succinylated peptides. ER, endoplasmic reticulum; PM, plasma membrane.
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