Abstract
Mitochondria require nicotinamide adenine dinucleotide (NAD+) to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD+ transporters have been identified in yeast and plants1,2, but their existence in mammals remains controversial3,4,5. Here we demonstrate that mammalian mitochondria can take up intact NAD+, and identify SLC25A51 (also known as MCART1)—an essential6,7 mitochondrial protein of previously unknown function—as a mammalian mitochondrial NAD+ transporter. Loss of SLC25A51 decreases mitochondrial—but not whole-cell—NAD+ content, impairs mitochondrial respiration, and blocks the uptake of NAD+ into isolated mitochondria. Conversely, overexpression of SLC25A51 or SLC25A52 (a nearly identical paralogue of SLC25A51) increases mitochondrial NAD+ levels and restores NAD+ uptake into yeast mitochondria lacking endogenous NAD+ transporters. Together, these findings identify SLC25A51 as a mammalian transporter capable of importing NAD+ into mitochondria.
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Data availability
The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. Source data are provided with this paper.
Code availability
No custom codes were used during this study. Mathematical calculations are described in the materials and methods section or by cited works.
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Acknowledgements
We thank all members of the Baur and Cambronne laboratories, V. Moiseenkova-Bell, A. Ellington, E. Marcotte, R. Goodman, I. Heiland, M. Whorton, E. Gouaux and J. Dixon for constructive discussions and suggestions; and M. Blair, Q. Chen, V. Annamalai, X. Yu, A. Slepian and CBRS UT Austin Proteomics Facility for technical support. This work was supported by grants from the National Institutes of Health (R01DK098656 to J.A.B., DP2GM126897 to X.A.C., TL1TR001880, T32AR53461 and F32HL145923 to T.S.L.) and the Norwegian Research Council (250395/F20 to M.Z.).
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T.S.L., X.A.C. and J.A.B. conceived and designed the overall study. T.S.L., J.M.E., M.-J.L., M.E.M., J.D.R., F.B.J., X.A.C. and J.A.B. contributed to the development of the hypotheses and experimental approaches. T.S.L., J.M.E., M.-J.L., M.N., F.R., M.R.M, C.P., M.R.B. and P.O. performed and analysed experiments. All authors contributed to the interpretation of experiments. T.S.L., X.A.C. and J.A.B. wrote the manuscript. J.M.E., M.-J.L., K.J. and M.Z. edited, and all authors reviewed the manuscript.
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J.D.R. is a co-founder of Toran Therapeutics. The remaining authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 SLC25A51 is a mitochondrial protein that affects cellular NAD+ distribution, proliferation, and metabolome profiles.
a, b, qPCR quantification of SLC25A51 mRNA expression in HEK 293T (n = 3) (a) and HeLa cells (n = 3) (b) expressing shRNA targeting SLC25A51. c, d, NAD+ content of isolated mitochondria (n = 3) (c) and whole cell lysates (n = 3) (d) from HeLa cells with stable shRNA knockdown of SLC25A51 (KD) and non-targeting control (Ctrl). e, Western blot confirming shRNA targeting murine Slc25a51 reduces SLC25A51 protein expression in cells transfected with cDNA encoding SLC25A51-FLAG. f, Mitochondrial free NAD+ levels in mouse embryonic stem cells expressing shRNA against Slc25a51 and non-targeting shRNA (shFF2), as measured with the mitochondrial cpVenus NAD+ biosensor (n = 3). g, qPCR quantification of SLC25A51 mRNA expression in HeLa cells transfected with siRNA targeting SLC25A51 (n = 3). h, Western blot confirming protein expression of Flag-tagged mitochondrial carriers. Controls include stable expression of the NAD+ biosensor (sensor) and anti-Tubulin for loading. i, Immunofluorescent detection of SLC25A51 and SLC25A52 subcellular localization. Cells were transiently transfected with cDNA encoding Flag-HA-tagged SLC25A51 or SLC25A52 and probed with anti-Flag and the mitochondrial marker, anti-MTC02. Scale bar: 10 μM, 2 μM on inset. Inset represents zoomed view of Flag localization and mitochondria. j–l, Proliferation of HAP1 SLC25A51-KO (n = 8) (j), HEK 293T SLC25A51 shRNA-knockdown (n = 8) (k), HeLa SLC25A51 shRNA-knockdown cells (n = 8) (l) and their respective controls. Proliferation was measured by CyQuant, a fluorescent DNA dye, at 0h and 96h after plating and expressed as fold change. m–o, qPCR quantification of SLC25A52 mRNA expression in HAP1 SLC25A51-KO (m), HEK 293T SLC25A51 shRNA-knockdown (n) and HeLa SLC25A51 shRNA-knockdown cells (n = 3) (o). p, Western blot of whole cell protein lysates from HAP1 wild type (WT) and SLC25A51 knockout (KO) cells confirming SLC25A51 loss. Loading control is total protein measured by Revert 700 Total Protein. q, r, Heat map of top 30 mitochondrial (q) and whole cell metabolites (r) that differ between HAP1 wild type and SLC25A51-KO cells (n = 3). Data represented as mean ± SEM. P values were determined by unpaired, two-tailed Student’s t-test (for two groups) or one-way ANOVA with multiple comparisons analysis using Dunnett’s method (for groups of three or more). *P < 0.05, and ***P < 0.001 vs control or WT (exact P values are provided in the source data).
Extended Data Fig. 2 NAD+ and SLC25A51 affect oxidative phosphorylation.
a, Respiration of isolated mitochondria from HEK 293T cells treated with either vehicle (Veh) or the NAMPT inhibitor FK866 to deplete mitochondrial NAD+. Mitochondria were treated with pyruvate and malate (state 2), then ADP was added to induce state 3 respiration. 1 mM NAD+ was added to test the ability of exogenous NAD+ to rescue respiration in the setting of mitochondria NAD+ depletion (Trace is representative of n = 4 independent experiments). P values were determined by two-way ANOVA with multiple comparisons analysis using the Sidak method. b, Oxygen consumption rate (OCR) was measured in SLC25A51 shRNA knockdown (KD) and control (Ctrl) HeLa cells using a Seahorse XF96e. Basal OCR was measured before the addition of treatments and maximal respiration was measured after the sequential addition of oligomycin (Oligo, ATP synthase inhibitor) and FCCP (uncoupler). Rotenone (Rot) and Antimycin A (AA) were added as a control to completely block mitochondrial oxygen consumption (n = 6). c, Respiration of isolated mitochondria from SLC25A51 knockdown HEK 293T cells. Mitochondria were treated with pyruvate/malate (state 2), and then ADP was added to induce state 3 respiration. Oligomycin was added to block ATP synthase-mediated respiration (n = 3 independent experiments). d, Mitochondria were isolated from HEK 293T control, SLC25A51 shRNA knockdown cells, and controls treated with FK866 to deplete mitochondrial NAD+. Mitochondrial oxygen consumption rate was measured after treatment with pyruvate/malate (state 2), ADP (state 3), and 1 mM NAD+ (n = 4 independent experiments). e, f, Mean volume per mitochondrial unit (e) and number of distinct mitochondria per cell (f) quantified from confocal image reconstructions of mitochondrial voxels in SLC25A51 shRNA knockdown (n = 31 cells) and control (n = 32 cells) HeLa cells. Data represented as mean ± SEM. P values were determined by unpaired, two-tailed Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001 vs vehicle or control; ###P < 0.001 vs state 3 (exact P values are provided in the source data).
Extended Data Fig. 3 Intact NAD+, but not nicotinamide or nicotinamide mononucleotide contributes to the mitochondrial NAD+ pool.
a, Mitochondrial NAD+ content was measured in isolated mitochondria from HeLa control (Ctrl) cells, control cells treated with FK866 (Ctrl+FK), and SLC25A51 shRNA-knockdown (KD) cells. NAD+ content of isolated mitochondria was determined before (untreated) and after a 40-min incubation with 1 mM NAD+ (n = 3 independent experiments). b, NAD+ levels in HEK 293T mitochondria incubated with 1 mM nicotinamide (NAM), 1 mM nicotinamide mononucleotide (NMN), or 1 mM NAD+ (n = 3 independent experiments). c, NAD+ uptake in NAD+-depleted mitochondria isolated from HEK 293T cells incubated with NAD+ ± 2 mM NAM or 2mM NMN (n = 4 independent experiments). d, Fractional labelling of mitochondrial NAD+ in HAP1 cells treated with isotopically double labelled NaR (n = 3 biological independent replicates). Data represented as mean ± SEM. P values were determined by unpaired, two-tailed Student’s t-test (for two groups) or one-way ANOVA with multiple comparisons analysis using Dunnett’s or Tukey’s method (for groups of three or more). *P < 0.05 and ***P < 0.001 vs untreated, vehicle, and wild-type M+0; #P < 0.05 vs wild-type M+1.
Extended Data Fig. 4 Generation and validation of yeast strains for testing mitochondrial NAD+ transport.
a, PCR genotyping to confirm double knockout gene deletion in BY4727 S. cerevisiae via antibiotic-resistance cassette replacement at the NDT1 and NDT2 loci. b, c, Deletion of the mitochondrial NAD+ carriers NDT1 and NDT2 in DKO strain phenocopied previously described growth defects on non-fermentative media (YP, 3% glycerol media)2, which was rescued by plasmid expression of NDT1. d, Western blot confirmed enrichment of mitochondrial markers (MTC02 and COXIV) and absence of cytoplasmic proteins (actin) or ER (SC2) in isolated mitochondria from yeast. e, RT-PCR confirmed ectopic expression from pRS415-SLC25A51 and pRS415-SLC25A52 in DKO strains.
Extended Data Fig. 5 Kinetics and selectivity of NAD+ transport by human SLC25A51 expressed in yeast mitochondria.
a–c,, Co-incubation with excess unlabelled NAD+ (n = 5 independent experiments for 1 mM NAD+) (a), supraphysiological levels of NMN (100 μM, n = 4 independent experiments; 500 μM, n = 5 independent experiments) (b), NADH (n = 3 independent experiments) with 3H-NAD+ to measure uptake competition in mitochondria from DKO yeast expressing SLC25A51 (c). d, Proportional relationship between integrated peak intensities from mass spectrometry of mitochondrial samples compared to a known meta data set of absolute protein abundances; used to quantitate SLC25A51 abundance in yeast samples. e, Uptake measured with indicated NAD+ concentrations; calculated from specific activity (n = 3 independent experiments, mean ± SEM). f, Lineweaver-Burk plot based on a nonlinear fit with datapoints overlaid (n = 3 independent experiments). P values were determined by two-way ANOVA with multiple comparisons analysis using Sidak’s method. *P < 0.05 and **P < 0.01 vs 100 μM cold NAD+.
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Luongo, T.S., Eller, J.M., Lu, MJ. et al. SLC25A51 is a mammalian mitochondrial NAD+ transporter. Nature 588, 174–179 (2020). https://doi.org/10.1038/s41586-020-2741-7
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DOI: https://doi.org/10.1038/s41586-020-2741-7
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