H+ transport is an integral function of the mitochondrial ADP/ATP carrier

Abstract

The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H⁺ transport. The AAC-mediated H⁺ current requires free fatty acids and resembles the H⁺ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H⁺ leak, but does not completely inhibit it. This suggests that the H⁺ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H⁺ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria.

Extended Data Figure 1 ∣. FA-dependent I_H in the IMM and plasma membrane.

a, Left panel: a diagram of patch-clamp recording from a vesicle of the whole IMM (mitoplast). After forming a gigaohm seal between the patch pipette and the mitoplast, the IMM patch under the pipette is broken by applying short pulses of high voltage (200–500 mV, 5-30 ms) combined with light suction to gain access into the mitoplast through the pipette. In this configuration, called the “whole-mitoplast” configuration, the interior of the mitoplast (mitochondrial matrix) is perfused with the pipette solution. The bath is also perfused to control the experimental solution on the cytosolic side of the IMM. The voltage across the IMM is set using the patch-clamp amplifier. Directions of currents flowing across the IMM: inward currents (flowing into the mitoplast) are negative, while outward currents are positive. Right panel: an example of a I_H current trace recorded in the whole-mitoplast mode. The voltage protocol used to induce the currents is shown above. All indicated voltages are within the mitochondrial matrix relative to the bath (cytosol). The voltage of the bath solution is defined to be zero. Baseline (zero current level) as well as negative (inward) and positive (outward) currents are indicated. b, I_H induced in a SM mitoplast by 1.5 μM (IMM, upper panel, n=11) or 15 μM (IMM, lower panel, n=3) AA. Voltage protocol is shown above the traces. Bath (cytosolic side of the IMM) and pipette (matrix side) pH are indicated in the pipette-mitoplast diagram. c, Same experiment performed in the plasma membrane (PM, n=4 at 1.5 μM, n=4 at 15 μM) of HEK293 cells. d, I_H densities in the IMM of SM and PM of HEK293 cells at 1.5 (n=11 for IMM, n=4 for PM) and 15 μM AA (n=3 for IMM, n=4 for PM). I_H measured at −160 mV. Data represent mean±SEM.

Extended Data Figure 2 ∣. UCP1-independent I_H in various mouse tissues.

a, I_H induced in mitoplasts of heart (n=6), liver (n=5), and brown fat (UCP1^−/− mice, n=6) by application of 1.5 μM AA on the cytosolic side of the IMM. Voltage protocol is shown above the traces. Bath and pipette pH are indicated in the pipette-mitoplast diagram. b, I_H induced in mitoplasts of skeletal muscle (SM) of wild-type (WT, n=7), UCP2^−/− (n=8), and UCP3^−/− (n=11) mice by application of 1.5 μM AA on the cytosolic side of the IMM. c, I_H densities in SM mitoplasts of WT (n=7), UCP2^−/−(n=8), and UCP3^−/− (n=11) mice, measured at −160 mV as in (b). Data represent mean±SEM. d, Representative SM mitochondrial I_H induced by 1.5 μM AA before (red) and after application of 1 mM GDP (blue) (n=4). e, Mitochondrial I_H recorded in the absence of added FA (control, black) was deactivated by addition of 10 mM MβCD to the bath (n=10).

Extended Data Figure 3 ∣. H⁺ selectivity of mitochondrial I_H.

a, Left panel, representative mitochondrial I_H recorded at ΔpH = 1 in response to the voltage step protocol indicated at the top (SM mitoplast, n=6); ΔV = 40 mV. A holding potential of −60 mV (close to the E_H) was selected to minimize H⁺ current and depletion of the proton buffer between applications of voltage steps. A zero current level is indicated by the red dotted line. Right panel, the I/V curve corresponding to the current traces in the left panel (SM mitoplast, n=6). Note the reversal potential. The pH values in the pipette and bath solutions are indicated on the diagram. b, Left panel, mitochondrial I_H recorded at ΔpH = 1.5 in response to the voltage step protocol indicated at the top of the panel (SM mitoplast, n=3); ΔV = 60 mV. Holding potential was −90 mV (close to the E_H). Right panel, the I/V curve corresponding to the current traces in the left panel (SM mitoplast, n=6). c, Left panel, mitochondrial I_H recorded at ΔpH = −0.5 in response to the voltage step protocol indicated at the top of the panel (SM mitoplast, n=4); ΔV = 40 mV. Holding potential was 0 mV. Right panel, the I/V curve corresponding to the current traces in the left panel. All currents were induced by 1.5 μM AA (SM mitoplast, n=6). d, I_H reversal potentials (V_rev) compared to Nernst H⁺ equilibrium potentials (E_H). Linear fitting of V_rev (red) and E_H at 24°C (black) vs. transmembrane ΔpH; pH 6/7, n=6; pH 6/7.5, n=3; pH 6.5/6, n=4. SM mitoplasts. Data represent mean±SEM.

Extended Data Figure 4 ∣. AAC-dependent and -independent currents induced by FA.

a, Current induced by 4 μM palmitic acid (PA, red) was inhibited by 1 μM CATR (blue). Control current is shown in black. Representative experiments performed in heart mitoplasts, n=4. b, The same experiment performed with 100 μM lauric acid (LA), n=5. c, Upper panel, currents induced by 2 2 μM of AA (green), PA (blue), and LA (red) in the same mitoplast. Control current is shown in black. Heart mitoplasts, n=4. Lower panel, mean I_H current densities at −160 mV induced by 2 μM of AA (n=6), PA (n=7), and LA (n=4) as in experiment shown in the upper panel. Heart mitoplasts. Data represent mean±SEM. d, Left panel: I_H induced by 2 μM AA (red) was inhibited by 4 μM BKA (blue). Control currents are shown in black. Representative experiment performed in a heart mitoplast (n=4). Right panel: inhibition of I_H induced by 2 μM AA in heart mitoplasts by 4 μM BKA. Remaining I_H measured at −160 mV is shown as a percentage of control, n=4. Paired t-test, two-tailed. Data represent mean±SEM. e, Current induced by 2 μM AA sulfonate before (red) and after (blue) addition of 1 μM CATR. Representative experiment performed in heart mitoplast. n=6. f, Current induced by 2 μM AA sulfonate before (red) and after (blue) addition of 50 μM mersalyl. Representative experiment performed in a heart mitoplast. n=4. g, Currents induced by 2 μM AA before (red) and after (blue) addition of 50 μM mersalyl. Note that only the outward current was inhibited. Representative experiment performed in a heart mitoplast. n=6. h, The outward current activated by 2 μM AA (red) is inhibited by 50 μM mersalyl (blue) and is next recovered by 1 mM DTT (green). Control current is shown in black. Heart mitoplasts, n= 4. i, Whole-mitoplast current before (control, black), after application of 2 μM AA (red), and upon washout of AA (blue). Heart mitoplasts, n=6. j, I_H induced by 2 μM AA (red) was inhibited by 1 μM CATR (blue). Control current is shown in black. Symmetrical pH 6.0. Heart mitoplasts, n=4. k, Inhibition of the inward I_H induced by 2 μM AA in SM, heart, liver, and kidney by 1 μM CATR. SM (n=22), heart (n=18), liver (n=4), and kidney (n=7) for both control and CATR treatment. Remaining inward current measured at −160 mV is shown as a percentage of control. Paired t test, two-tailed. Data represent mean±SEM. l, Inhibition of the outward current induced by 2 μM AA in SM, heart, liver, and kidney by 1 μM CATR. Remaining outward current measured at +100 mV is shown as a percentage of control. SM (n=21), heart (n=17), liver (n=4), and kidney (n=7) for both control and CATR treatment. Paired t-test, two-tailed. Data represent mean±SEM.

Extended Data Figure 5 ∣. FA-dependent I_H via AAC is potentiated by oxidation.

a,c,e, I_H activated by 2 μM AA (red) was then potentiated by oxidizers 250 μM tBHP, 100 μM 4-HNE, or 20 μM TBT (blue). I_H potentiated by oxidizers was inhibited by CATR (green). Control current is shown in black. Bar graphs show ratio of I_H amplitudes at −160 mV before and after addition of oxidizer. Heart mitoplasts. Note that TBT and 4-HNE, but not tBHP, inhibited the AAC-independent outward current observed at positive membrane potentials. Panel (a) n=4, panel (c) n=3, and panel (e) n=5 for all experimental conditions. Paired t-test, two-tailed. Data represent mean±SEM. b,d,f, Currents before (control, black) and after (red) application of 250 μM tBHP (n=3), 20 μM TBT (n=3), and 100 μM 4-HNE (n=3).

Extended Data Figure 6 ∣. FA-dependent currents in AAC1 knockout and AAC2 hypomorphic mice.

a and b, Representative currents induced by 2 μM AA in WT and AAC2 hypomorphic mitoplasts of heart (n=9 for WT and n=9 for hypo) (a) and kidney (n=4 for WT and n=5 for hypo) (b). Right panels: I_H current densities at −160 mV for WT (n=10 for heart and n=5 for kidney) and AAC2 hypomorphic mitoplasts (n=10 for heart and n=6 for kidney). Data are mean±SEM. c-e, Densities of the outward current measured at +100 mV for WT and AAC1^−/− mitoplasts of heart (n=14 for WT and n=10 for AAC1^−/−), SM (n=21 for WT and n=12 for AAC1^−/−), and kidney (n=5 for WT and n=6 for AAC1^−/−). Mann-Whitney test, two-tailed. Data are mean±SEM. f and g, Densities of the outward current measured at +100 mV for WT (n=9 for heart and n=5 for kidney) and AAC2 hypomorphic (n=10 for heart and n=5 for kidney) mitoplasts of heart and kidney. Mann-Whitney test, two-tailed. Data are mean±SEM. h, Inhibition of the outward current induced by 2 μM AA in AAC1^−/− heart mitoplasts by 1 μM CATR (n=5, control and CATR). Remaining outward current measured at +100 mV is shown as a percentage of control. Paired t-test, two-tailed. Data are mean±SEM. i, Left panel: inhibition of inward I_H induced by 2 μM AA in AAC1^−/− SM mitoplasts by 1 μM CATR (n=10, control and CATR). Remaining I_H measured at −160 mV is shown as a percentage of control. Right panel: inhibition of the outward current induced by 2 μM AA in in AAC1^−/− SM mitoplasts by 1 μM CATR (n=9, control and CATR). Remaining current measured at +100 mV is shown as a percentage of control. Paired t-test, two-tailed. Data are mean±SEM. j, Two representative experiments in which the I_H induced by 2μM AA in AAC1^−/− mitoplasts of SM was the smallest (left panel, n=4) and the largest (right panel, n=3). I_H induced by 2 μM AA (red) was inhibited by 1 μM CATR (blue). Control current is shown in black.

Extended Data Figure 7 ∣. Interaction of FA anions with AAC.

a, I_H induced by 2 μM AA (red) was inhibited by 66±2% (n=4, SM mitoplasts) by 5 μM AA-sulf (blue). Data are mean±SEM. b, Current induced by 5 μM AA-sulf (left panel, red) or 1 μM AA (right panel, red) was inhibited by 1 μM CATR (blue). Control currents are shown in black. Smaller [AA] was used to induce comparable currents with AA-sulf. Heart mitoplasts, n=4. c and d, Currents recorded before (control, black) and after addition of 5 μM AA-sulf (c) or 10 mM C6-sulf (d) to bath (red). Brown fat mitoplast (UCP1, left panel), heart mitoplast (AAC, right panel), n=4 for each. Currents were measured at pH 6.0 to inhibit the production of FA by phospholipase A2 (PLA2) associated with the brown fat IMM and ensure that UCP1 currents were activated by exogenously applied FA anions only. e, Current before (control, black) and after (red) application of 50 mM C6-sulf to the bath. Pipette solution contained 50 mM C6-sulf. Symmetrical pH 6.0. Heart mitoplasts, n=3. f, Current before (control, black) and after (red) application of 5 μM AA-sulf to the bath. Pipette solution contained 10 μM AA-sulf. Bath AA-sulf was kept at 5 μM because higher concentrations disrupted the IMM. Symmetrical pH 6.0. Heart mitoplasts, n=3. g, Proposed model of FA-dependent I_H via AAC. Without FA, AAC is impermeable for H⁺ (1). When FA binds in the AAC translocation pathway, its protonatable headgroup enables H⁺ binding and transport (2). FA can activate I_H with AAC in either the c- or m-state (2 and 3). Because the SBS is positively charged and retains its structure with c–m conformational change, the negatively charged head of FA is likely to interact with the SBS, while the hydrophobic carbon tail may protrude into the membrane and/or be stabilized by hydrophobic interactions within AAC (2 and 3).

Extended Data Figure 8 ∣. Adenine nucleotide exchange by AAC.

a, The alternating access mechanism of adenine nucleotide transport by AAC. AAC is shown in green, and its substrate-binding site (SBS, overall positively charged) located in the middle of the membrane is shown in blue. Cytosolic ADP binds to AAC in the c-state (1). AAC transitions to the m-state, and ADP is released into the matrix (2 and 3). Matrix ATP binds to AAC in the m-state (4). AAC transitions to the c-state, and ATP is released into the cytosol (5). b, AAC current activated by 1 mM ADP (red) is inhibited by 1μM CATR (blue). Pipette solution contained and 1 mM ATP. Heart mitoplast, n=3. Control trace is in black. c, Inhibition of the inward ADP/ATP exchange current via AAC by 1 μM CATR. Heart mitoplast, n=6 (control and CATR treatment). Paired t-test, two-tailed. Data are mean±SEM. Remaining inward current measured at −160 mV is shown as a percentage of control. See also Fig. 3a. d, Inhibition of the outward ATP/ADP exchange current via AAC by 1 μM CATR. Heart mitoplast, n=5 (control and CATR treatment). Paired t-test, two-tailed. Data are mean±SEM. Remaining outward current measured at −100 mV is shown as a percentage of control. See also Fig. 3b. e, Inhibition of the inward I_H induced by 2 μM AA by 1 μM CATR after ADP pre-treatment. Heart mitoplast, n=7 (control and CATR treatment). Paired t-test, two-tailed. Data are mean±SEM. Remaining I_H measured at −160 mV is shown as a percentage of control. See also Fig. 3e. f, Current before (control, black) and after (red) addition of 2 μM AA to bath. Subsequent addition of 1 μM CATR (blue) inhibited I_H. Pipette solution contained 4 μM AA. Heart mitoplast, n=4. g, Control current (black) and current after addition of 1 mM ADP to the bath solution (red). AA (2 μM) was added to the bath solution at the end of experiment (blue). Pipette solution contained 4 μM AA. Heart mitoplasts, n=4.

Extended Data Figure 9 ∣. Regulation of FA-dependent I_H by nucleotides.

a, Explanation of transient I_H inhibition by cytosolic adenine nucleotides. AAC in c-state, with FA anion in the translocation pathway, mediates I_H (1). Cytosolic ADP³⁻ binds in c-state and expels FA anion/blocks translocation pathway, leading to I_H inhibition (2). Upon AAC conformation change, ADP dissociates into matrix (pipette) solution (3). FA anion re-associates with AAC in m-state, restoring I_H (4). Cytosolic ADP cannot inhibit I_H while AAC is in m-state (5). See also Fig. 4a. b, Proposed mechanism of I_H inhibition by adenine nucleotide exchange. AAC in c-state, with FA anion in the translocation pathway, mediates I_H (1). Cytosolic ADP³⁻ binds in c-state and expels FA anion/blocks translocation pathway, leading to I_H inhibition (2). The resultant continuous exchange of cytosolic and matrix adenine nucleotides inhibits FA anion binding and I_H (3, 4, and 5). ATP (and not ADP) is shown as a matrix adenine nucleotide to reflect physiological conditions. See also Fig. 4b. c, Remaining I_H after inhibition by different concentrations of ADP applied to both sides of the IMM to induce continuous adenine nucleotide exchange via AAC as in (e). ADP/ADP exchange was used to avoid contaminating I_H with ADP/ATP exchange current. Heart and SM mitoplasts, n=5 (control and 10 μM ADP), n=8 (control and 100 μM ADP, n=9 (control and 1 mM ADP). Data are mean±SEM. d, I_H via UCP1 is inhibited by 100 μM Mg²⁺-free ADP (upper panel, n=5) and 1 mM Mg²⁺-free ADP (lower panel, n=3). I_H activated by 2 μM AA is shown before (red) and after inhibition by ADP (blue). In the beginning of the experiment, before the application of AA, the endogenous membrane FA were removed by a 30-40s pre-treatment with 10 mM MβCD (black, control). All recording solutions contained 1 μM CATR to reduce AAC contribution to the I_H measured. Pipette solution contained either 100 μM ADP (upper panel) or 1 mM ADP (lower panel) to match the recording conditions for AAC (e). Brown fat mitoplasts. e, I_H via AAC is inhibited by 100 μM Mg²⁺-free ADP (upper panel, n=8) and 1 mM Mg²⁺-free ADP (lower panel, n=8). I_H activated by 2 μM AA is shown before (red) and after inhibition by ADP (blue). Pipette solution contained either 100 μM ADP (upper panel) or 1 mM ADP (lower panel) to achieve symmetrical [ADP] on both side of IMM. Heart mitoplasts. f, Mean densities of I_H via UCP1 (dark grey) and AAC (light grey) in control (brown fat, n=11 and heart, n=9) and in the presence of 100 μM (brown fat, n=5 and heart, n=8) and 1 mM ADP (brown fat, n=3 and heart, n=8) on both sides of the IMM. I_H amplitudes were measured at −160 mV. The same data as in Fig. 4e. Data represent mean±SEM.

Extended Data Figure 10 ∣. Phenotypes associated with AAC deficiency.

a, Representative OCRs of isolated heart mitochondria from WT (left panel, n=3 wells) and AAC1^−/− mice (right panel, n=4 wells). As indicated by the arrows, first oligomycin and then either palmitic acid (PA: 50 μM, light orange and 100 μM, dark orange) or buffer (black) were added, following by FCCP and rotenone. Higher PA concentrations were used than those for electrophysiological experiments, because in suspensions of isolated mitochondria and in the presence of albumin, the effective concentration of PA is significantly lower. FCCP-induced uncoupled respiration in WT and AAC1^−/− mitochondria validated their respiration capacity. Data represent mean±SEM. This experiment was repeated with independent mitochondrial isolations in WT (n=4) and AAC1^−/− (n=3) with the same results. b, Basal OCR of isolated heart mitochondria of WT (n=24 wells) and AAC1^−/− (n= 18 wells) mice. Mann-Whitney test, two-tailed. Data represent mean±SEM. c, Representative immunoblots in WT (n=5) and DKO (n=7) C2C12 cells for: NDUFB8 (complex I, CI), SDHA (complex II, CII), core 2 subunit (complex III, CIII), CIV-I subunit (complex IV, CIV), and ATP5A (complex V, CV), TOM20, and the loading control (plasma membrane Na⁺/K⁺ ATPase). For gel source data see Supplementary Figure 1. d, Basal and ADP-stimulated OCR of mitochondria from WT (basal, ADP 100 μM, and ADP 200 μM, n=20) and DKO (n=16 for basal, n=16 for ADP 100 μM, and n=17 for ADP 200 μM) C2C12 cells. Mann-Whitney test, two-tailed. Data represent mean±SEM. e, Representative confocal micrographs of WT (upper panels, n=45 cells) and DKO (lower panels, n=45 cells) C2C12 cells immunolabeled with TOM20 (green) and tubulin (red) antibodies. Insets show magnified area from the same images. f, Mitochondrial biomass per cell in WT and DKO C2C12 cells, calculated as a ratio between TOM20 signal and the total area of the cell, n=45 per each group. Data represent mean±SEM. g, Comparison of a ratio between mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) from WT (n=6) and DKO (n=6) C2C12 cells. Data represent mean±SEM. h, Kinetic study of ECAR in WT (n=22) and DKO (n=22) C2C12 cells under basal conditions and upon addition of oligomycin into the respiration medium. Note that inhibition of mitochondrial ATP production in DKO cells with oligomycin did not affect ECAR, whereas in WT cells, oligomycin potently stimulated ECAR. Data represent mean±SEM.

Figure 1 ∣. Pharmacological and biophysical properties of I_H.

a, I_H induced by 2 μM AA (red) was inhibited by 1 μM CATR (blue) in SM (n=22), heart (n=18), kidney (n=4), and liver (n=7) mitoplasts. Right panel: I_H current densities at −160 mV in SM (n=56), heart (n=67), kidney (n=8), and liver (n=13) . Data represent mean±SEM. b, Representative recording from SM (upper panel, n=5) and heart (lower panel, n=4) mitoplasts pre-treated with 50 μM mersalyl. I_H induced by 2 μM AA (red) was inhibited by 1 μM CATR (blue). c, UCP1-dependent I_H induced by 2 μM AA (red) in brown fat mitoplast (n=3) was inhibited by 1 mM GDP (blue). d, Densities of AAC and UCP1 I_H induced by 2 μM AA at −160 mV in SM (n=56) and brown fat (n=3). Data represent mean±SEM. e, Representative I_H induced by 2 μM AA at different membrane voltages. Heart mitoplasts, n=4. f, Current-voltage (I/V) curve of I_H based on data in (e). Data are mean±SEM.

Figure 2 ∣. AAC is required for I_H.

a-c, Representative currents induced by 2 μM AA in WT and AAC1^−/− mitoplasts of heart (a), SM (b), and kidney (c). Right panels: I_H current densities at −160 mV for WT and AAC1^−/− mitoplasts. All AAC1^−/− experiments used the same WT control as in Fig. 1a. Heart: WT, n=67 and AAC1^−/−, n=11. SM: WT, n=56 and AAC1^−/−, n=14. Kidney: WT, n=13 and AAC1^−/−, n=6. Mann-Whitney test, two-tailed. Data are mean±SEM.

Figure 3 ∣. Adenine nucleotide transport by AAC.

a, Left panel: AAC current before (control, black) and after (red) addition of 5 mM ADP to the bath solution. Pipette solution contained 5 mM ATP. Subsequent addition of 5 μM CATR (blue) inhibited ADP/ATP exchange. Right panel: The same experiment performed in AAC1^−/− mitoplasts. Heart mitoplasts, n=7 (WT), and n=4 (AAC1^−/−). b, AAC current before (control, black) and after (red) addition of 5 mM ATP to the bath solution. Pipette solution contained 5 mM ADP. Subsequent addition of 5 μM CATR (blue) inhibited ADP/ATP exchange. Heart mitoplasts, n=5. c, Current before (control, black) and after (red) addition of 5 mM ADP to the bath solution. Pipette solution contained 5 mM ADP. Heart mitoplasts, n=3. d, Densities of the ADP/ATP exchange current in WT (n=7) and AAC1^−/− (n=4) heart mitoplasts, measured at −160 mV in (a). Mann-Whitney test, two-tailed. Data represent mean±SEM. e, I_H activated by 2 μM AA before (red) and after (blue) addition of 1 μM CATR to bath (SM mitoplast). The mitoplast was pretreated with 1 mM ADP just before AA application. Heart and SM mitoplasts, n=7.

Figure 4 ∣. Nucleotide exchange negatively regulates I_H.

a, Left panel: I_H induced by 2 μM AA (1), followed by a transient inhibition by 1 mM of bath ADP (2), and subsequent recovery (3). SM mitoplast. Right panel: I_H time course of left panel. I_H was measured at −160 mV. The experiment was repeated with the similar result (n=7) in heart and SM mitoplasts. b, Left panel: I_H activated by 2 μM AA (red) was inhibited by addition of 1 mM ADP to bath. Pipette solution contained 1 mM ADP. Heart mitoplast. Right panel: I_H time course of the left panel. The experiment was repeated with the similar result (n=9) in heart and SM mitoplasts. c, ADP inhibition of I_H in points 2 and 3 as in (a). Heart and SM mitoplasts, n=7. Paired t-test, two-tailed. Data are mean±SEM. d, ADP inhibition of I_H in point 2 as in (b). Heart and SM mitoplasts, n=9. Paired t-test, two-tailed. Data are mean±SEM. e, Mean densities of I_H via UCP1 (dark grey) and AAC (light grey) in control (n=11, UCP1; n=9, AAC) and in the presence of 100 μM (n=5, UCP1; n=8, AAC) and 1 mM ADP (n=3, UCP1; n=8, AAC). I_H was measured as in Extended Data Fig. 9e and f. Brown fat and heart mitoplasts. Data are mean±SEM.

Figure 5 ∣. Mitochondrial uncoupling requires AAC.

a, OCR of isolated heart mitochondria of WT (left panel) and AAC1^−/− mice (right panel) normalized to the basal value. Arrows indicate addition of oligomycin (Oligo) and either palmitic acid (PA: 50 μM, light orange, n=5 for WT, n=3 for AAC1^−/−; and 100 μM, dark orange, n=4 for WT, n=3 for AAC1^−/−) or buffer (black, n=5 for WT, n=3 for AAC1^−/−). Each n corresponds to independent mitochondrial isolations (also see Methods). The effect of the PA was recorded upon addition (t0) and 30 min later (t30). Paired t-test, two-tailed. **no FA (P=0.0009) vs 50 μM PA; #no FA vs 100 μM (P=0.0183). Data represent mean±SEM. b, OCR of mitochondria isolated from WT (black) and DKO (grey) C2C12 cells before (basal, oligomycin added; n=50 for WT, and n=47 for DKO) and after addition of gramicidin A (n=40 for WT and n=30 for DKO). Each n corresponds to individual respiration wells. Mann-Whitney test, two-tailed. Data represent mean±SEM. c, OCR change in WT (grey, n=20 wells) and DKO (white, n=16 wells) C2C12 isolated mitochondria upon sequential addition of increasing concentrations of PA, and FCCP. All OCR changes were normalized per mean basal OCR (before FA addition). Mann-Whitney test, two-tailed. ***, P=0.0021; ****, P>0.0001 compared to basal. Data represent mean±SEM. d, OCR of WT (black, n=22 wells) and DKO (grey, n=22 wells) C2C12 cells before (basal) and after addition of oligomycin. Mann-Whitney test, two-tailed. Data represent mean±SEM. e, OCR change in WT (grey, n=20 wells) and DKO (white, n=19 wells) C2C12 cells after sequential addition of increasing concentrations of PA, and FCCP. All OCR changes were normalized per the mean oligomycin OCR (before FA addition). Mann-Whitney test, two-tailed. ****, P>0.0001 compared to oligomycin. Data represent mean±SEM. The growth medium for WT and DKO cell contained 25 mM glucose.