Mitochondrial dysfunction by TFAM depletion disrupts self-renewal and lineage differentiation of human PSCs by affecting cell proliferation and YAP response.

Genetic mitochondrial dysfunction is frequently associated with various embryonic developmental defects. However, how mitochondria contribute to early development and cell fate determination is poorly studied, especially in humans. Using human pluripotent stem cells (hPSCs), we established a Dox-induced knockout model with mitochondrial dysfunction and evaluated the effect of mitochondrial dysfunction on human pluripotency maintenance and lineage differentiation. The nucleus-encoded gene TFAM (transcription factor A, mitochondrial), essential for mitochondrial gene transcription and mitochondrial DNA replication, is targeted to construct the mitochondrial dysfunction model. The hPSCs with TFAM depletion exhibit the decrease of mtDNA level and oxidative respiration efficiency, representing a typical mitochondrial dysfunction phenotype. Mitochondrial dysfunction leads to impaired self-renewal in hPSCs due to proliferation arrest. Although the mitochondrial dysfunction does not affect pluripotent gene expression, it results in a severe defect in lineage differentiation. Further study in mesoderm differentiation reveals that mitochondrial dysfunction causes proliferation disability and YAP nuclear translocalization and thus together blocks mesoderm lineage differentiation. These findings provide new insights into understanding the mitochondrial function in human pluripotency maintenance and mesoderm differentiation.

Retinal pigment epithelium-specific CLIC4 mutant is a mouse model of dry age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Dry AMD has unclear etiology and no treatment. Lipid-rich drusen are the hallmark of dry AMD. An AMD mouse model and insights into drusenogenesis are keys to better understanding of this disease. Chloride intracellular channel 4 (CLIC4) is a pleomorphic protein regulating diverse biological functions. Here we show that retinal pigment epithelium (RPE)-specific Clic4 knockout mice exhibit a full spectrum of functional and pathological hallmarks of dry AMD. Multidisciplinary longitudinal studies of disease progression in these mice support a mechanistic model that links RPE cell-autonomous aberrant lipid metabolism and transport to drusen formation.

Characterization of Poldip2 knockout mice: Avoiding incorrect gene targeting.

POLDIP2 is a multifunctional protein whose roles are only partially understood. Our laboratory previously reported physiological studies performed using a mouse gene trap model, which suffered from three limitations: perinatal lethality in homozygotes, constitutive Poldip2 inactivation and inadvertent downregulation of the adjacent Tmem199 gene. To overcome these limitations, we developed a new conditional floxed Poldip2 model. The first part of the present study shows that our initial floxed mice were affected by an unexpected mutation, which was not readily detected by Southern blotting and traditional PCR. It consisted of a 305 kb duplication around Poldip2 with retention of the wild type allele and could be traced back to the original targeted ES cell clone. We offer simple suggestions to rapidly detect similar accidents, which may affect genome editing using both traditional and CRISPR-based methods. In the second part of the present study, correctly targeted floxed Poldip2 mice were generated and used to produce a new constitutive knockout line by crossing with a Cre deleter. In contrast to the gene trap model, many homozygous knockout mice were viable, in spite of having no POLDIP2 expression. To further characterize the effects of Poldip2 ablation in the vasculature, RNA-seq and RT-qPCR experiments were performed in constitutive knockout arteries. Results show that POLDIP2 inactivation affects multiple cellular processes and provide new opportunities for future in-depth study of its functions.

FARS2 deficiency in Drosophila reveals the developmental delay and seizure manifested by aberrant mitochondrial tRNA metabolism.

Mutations in genes encoding mitochondrial aminoacyl-tRNA synthetases are linked to diverse diseases. However, the precise mechanisms by which these mutations affect mitochondrial function and disease development are not fully understood. Here, we develop a Drosophila model to study the function of dFARS2, the Drosophila homologue of the mitochondrial phenylalanyl-tRNA synthetase, and further characterize human disease-associated FARS2 variants. Inactivation of dFARS2 in Drosophila leads to developmental delay and seizure. Biochemical studies reveal that dFARS2 is required for mitochondrial tRNA aminoacylation, mitochondrial protein stability, and assembly and enzyme activities of OXPHOS complexes. Interestingly, by modeling FARS2 mutations associated with human disease in Drosophila, we provide evidence that expression of two human FARS2 variants, p.G309S and p.D142Y, induces seizure behaviors and locomotion defects, respectively. Together, our results not only show the relationship between dysfunction of mitochondrial aminoacylation system and pathologies, but also illustrate the application of Drosophila model for functional analysis of human disease-causing variants.

Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome.

So-called ρ0 cells lack mitochondrial DNA and are therefore incapable of aerobic ATP synthesis. How cells adapt to survive ablation of oxidative phosphorylation remains poorly understood. Complexome profiling analysis of ρ0 cells covered 1,002 mitochondrial proteins and revealed changes in abundance and organization of numerous multiprotein complexes including previously not described assemblies. Beyond multiple subassemblies of complexes that would normally contain components encoded by mitochondrial DNA, we observed widespread reorganization of the complexome. This included distinct changes in the expression pattern of adenine nucleotide carrier isoforms, other mitochondrial transporters, and components of the protein import machinery. Remarkably, ablation of mitochondrial DNA hardly affected the complexes organizing cristae junctions indicating that the altered cristae morphology in ρ0 mitochondria predominantly resulted from the loss of complex V dimers required to impose narrow curvatures to the inner membrane. Our data provide a comprehensive resource for in-depth analysis of remodeling of the mitochondrial complexome in response to respiratory deficiency.

Mitofusin 2 Deficiency Causes Pro-Inflammatory Effects in Human Primary Macrophages.

Mitofusin 2 (MFN2) is a mitochondrial outer membrane GTPase, which modulates mitochondrial fusion and affects the interaction between endoplasmic reticulum and mitochondria. Here, we explored how MFN2 influences mitochondrial functions and inflammatory responses towards zymosan in primary human macrophages. A knockdown of MFN2 by small interfering RNA decreased mitochondrial respiration without attenuating mitochondrial membrane potential and reduced interactions between endoplasmic reticulum and mitochondria. A MFN2 deficiency potentiated zymosan-elicited inflammatory responses of human primary macrophages, such as expression and secretion of pro-inflammatory cytokines interleukin-1ß, -6, -8 and tumor necrosis factor α, as well as induction of cyclooxygenase 2 and prostaglandin E2 synthesis. MFN2 silencing also increased zymosan-induced nuclear factor kappa-light-chain-enhancer of activated B cells and mitogen-activated protein kinases inflammatory signal transduction, without affecting mitochondrial reactive oxygen species production. Mechanistic studies revealed that MFN2 deficiency enhanced the toll-like receptor 2-dependent branch of zymosan-triggered responses upstream of inhibitor of κB kinase. This was associated with elevated, cytosolic expression of interleukin-1 receptor-associated kinase 4 in MFN2-deficient cells. Our data suggest pro-inflammatory effects of MFN2 deficiency in human macrophages.

Loss of the mitochondrial phosphate carrier SLC25A3 induces remodeling of the cardiac mitochondrial protein acylome.

Mitochondria are recognized as signaling organelles, because under stress, mitochondria can trigger various signaling pathways to coordinate the cell's response. The specific pathway(s) engaged by mitochondria in response to mitochondrial energy defects in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. Mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased posttranslational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3-deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel cross talk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the energy production machinery can have an expanded impact on global mitochondrial function.

Ist2 recruits the lipid transporters Osh6/7 to ER-PM contacts to maintain phospholipid metabolism.

ER-plasma membrane (PM) contacts are proposed to be held together by distinct families of tethering proteins, which in yeast include the VAP homologues Scs2/22, the extended-synaptotagmin homologues Tcb1/2/3, and the TMEM16 homologue Ist2. It is unclear whether these tethers act redundantly or whether individual tethers have specific functions at contacts. Here, we show that Ist2 directly recruits the phosphatidylserine (PS) transport proteins and ORP family members Osh6 and Osh7 to ER-PM contacts through a binding site located in Ist2's disordered C-terminal tethering region. This interaction is required for phosphatidylethanolamine (PE) production by the PS decarboxylase Psd2, whereby PS transported from the ER to the PM by Osh6/7 is endocytosed to the site of Psd2 in endosomes/Golgi/vacuoles. This role for Ist2 and Osh6/7 in nonvesicular PS transport is specific, as other tethers/transport proteins do not compensate. Thus, we identify a molecular link between the ORP and TMEM16 families and a role for endocytosis of PS in PE synthesis.

Loss of mitochondrial transcription factor A in neural stem cells leads to immature brain development and triggers the activation of the integral stress response in vivo.

Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.

HSP60 reduction protects against diet-induced obesity by modulating energy metabolism in adipose tissue.

OBJECTIVE: Insulin regulates mitochondrial function, thereby propagating an efficient metabolism. Conversely, diabetes and insulin resistance are linked to mitochondrial dysfunction with a decreased expression of the mitochondrial chaperone HSP60. The aim of this investigation was to determine the effect of a reduced HSP60 expression on the development of obesity and insulin resistance. METHODS: Control and heterozygous whole-body HSP60 knockout (Hsp60+/-) mice were fed a high-fat diet (HFD, 60% calories from fat) for 16 weeks and subjected to extensive metabolic phenotyping. To understand the effect of HSP60 on white adipose tissue, microarray analysis of gonadal WAT was performed, ex vivo experiments were performed, and a lentiviral knockdown of HSP60 in 3T3-L1 cells was conducted to gain detailed insights into the effect of reduced HSP60 levels on adipocyte homeostasis. RESULTS: Male Hsp60+/- mice exhibited lower body weight with lower fat mass. These mice exhibited improved insulin sensitivity compared to control, as assessed by Matsuda Index and HOMA-IR. Accordingly, insulin levels were significantly reduced in Hsp60+/- mice in a glucose tolerance test. However, Hsp60+/- mice exhibited an altered adipose tissue metabolism with elevated insulin-independent glucose uptake, adipocyte hyperplasia in the presence of mitochondrial dysfunction, altered autophagy, and local insulin resistance. CONCLUSIONS: We discovered that the reduction of HSP60 in mice predominantly affects adipose tissue homeostasis, leading to beneficial alterations in body weight, body composition, and adipocyte morphology, albeit exhibiting local insulin resistance.

Aberrant activity of mitochondrial NCLX is linked to impaired synaptic transmission and is associated with mental retardation.

Calcium dynamics control synaptic transmission. Calcium triggers synaptic vesicle fusion, determines release probability, modulates vesicle recycling, participates in long-term plasticity and regulates cellular metabolism. Mitochondria, the main source of cellular energy, serve as calcium signaling hubs. Mitochondrial calcium transients are primarily determined by the balance between calcium influx, mediated by the mitochondrial calcium uniporter (MCU), and calcium efflux through the sodium/lithium/calcium exchanger (NCLX). We identified a human recessive missense SLC8B1 variant that impairs NCLX activity and is associated with severe mental retardation. On this basis, we examined the effect of deleting NCLX in mice on mitochondrial and synaptic calcium homeostasis, synaptic activity, and plasticity. Neuronal mitochondria exhibited basal calcium overload, membrane depolarization, and a reduction in the amplitude and rate of calcium influx and efflux. We observed smaller cytoplasmic calcium transients in the presynaptic terminals of NCLX-KO neurons, leading to a lower probability of release and weaker transmission. In agreement, synaptic facilitation in NCLX-KO hippocampal slices was enhanced. Importantly, deletion of NCLX abolished long term potentiation of Schaffer collateral synapses. Our results show that NCLX controls presynaptic calcium transients that are crucial for defining synaptic strength as well as short- and long-term plasticity, key elements of learning and memory processes.

MTM1 plays an important role in the regulation of zinc tolerance in Saccharomyces cerevisiae.

BACKGROUND: Acquisition and distribution of zinc supports a number of biological processes. Various molecular factors are involved in zinc metabolism but not fully explored. BASIC PROCEDURES: Spontaneous mutants were generated in yeast with excess zinc culture followed by whole genome DNA sequencing to discover zinc metabolism related genes by bioinformatics. An identified mutant was characterized through metallomic and molecular biology methods. MAIN FINDINGS: Here we reported that MTM1 knockout cells displayed much stronger zinc tolerance than wild type cells on SC medium when exposed to excess zinc. Zn accumulation of mtm1Δ cells was dramatically decreased compared to wild type cells under excessive zinc condition due to MTM1 deletion reduced zinc uptake. ZRC1 mRNA level of mtm1Δ cells was significantly higher than that in the wild-type strain leading to increased vacuolar zinc accumulations in mtm1Δ cells. The mRNA levels of ZRT1 and ZAP1 decreased in mtm1Δ cells contributing to less Zn uptake. The zrc1Δmtm1Δ double knockout strain exhibited Zn sensitivity. MTM1 knockout did not afford resistance to excess zinc through an effect mediated through an influence on levels of ROS. Superoxide dismutase 2 (Sod2p) activity in mtm1Δ cells was severely impaired and not restored through Zn supplementation. Meanwhile, additional Zn showed no significant effect on the localization and expression of Mtm1p. PRINCIPAL CONCLUSIONS: Our study reveals the MTM1 gene plays an important role in the regulation of zinc homeostasis in yeast cells via changing zinc uptake and distribution. This discovery provides new insights for better understanding biochemical communication between vacuole and mitochondrial in relation to zinc-metabolism.

Identification of highest neurotoxic amyloid-ß plaque type showing reduced contact with astrocytes.

Amyloid-ß (Aß) plaques are strongly associated with the development of Alzheimer's disease (AD). However, it remains unclear how morphological differences in Aß plaques determine the pathogenesis of Aß. Here, we categorized Aß plaques into four types based on the macroscopic features of the dense core, and found that the Aß-plaque subtype containing a larger dense core showed the strongest association with neuritic dystrophy. Astrocytes dominantly accumulated toward these expanded/dense-core-containing Aß plaques. Previously, we indicated that deletion of the mitochondrial ubiquitin ligase MITOL/MARCH5 triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aß pathology. In this study, MITOL deficiency accelerated the formation of expanded/dense-core-containing Aß plaques, which showed reduced contacts with astrocytes, but not microglia. Our findings suggest that expanded/dense-core-containing Aß-plaque formation enhanced by the alteration of mitochondrial function robustly contributes to the exacerbation of Aß neuropathology, at least in part, through the reduced contacts between Aß plaques and astrocytes.

Choline restores respiration in Psd1-deficient yeast by replenishing mitochondrial phosphatidylethanolamine.

Phosphatidylethanolamine (PE) is essential for mitochondrial respiration in yeast, Saccharomyces cerevisiae, whereas the most abundant mitochondrial phospholipid, phosphatidylcholine (PC), is largely dispensable. Surprisingly, choline (Cho), which is a biosynthetic precursor of PC, has been shown to rescue the respiratory growth of mitochondrial PE-deficient yeast; however, the mechanism underlying this rescue has remained unknown. Using a combination of yeast genetics, lipid biochemistry, and cell biological approaches, we uncover the mechanism by showing that Cho rescues mitochondrial respiration by partially replenishing mitochondrial PE levels in yeast cells lacking the mitochondrial PE-biosynthetic enzyme Psd1. This rescue is dependent on the conversion of Cho to PC via the Kennedy pathway as well as on Psd2, an enzyme catalyzing PE biosynthesis in the endosome. Metabolic labeling experiments reveal that in the absence of exogenously supplied Cho, PE biosynthesized via Psd2 is mostly directed to the methylation pathway for PC biosynthesis and is unavailable for replenishing mitochondrial PE in Psd1-deleted cells. In this setting, stimulating the Kennedy pathway for PC biosynthesis by Cho spares Psd2-synthesized PE from the methylation pathway and redirects it to the mitochondria. Cho-mediated elevation in mitochondrial PE is dependent on Vps39, which has been recently implicated in PE trafficking to the mitochondria. Accordingly, epistasis experiments placed Vps39 downstream of Psd2 in Cho-based rescue. Our work, thus, provides a mechanism of Cho-based rescue of mitochondrial PE deficiency and uncovers an intricate interorganelle phospholipid regulatory network that maintains mitochondrial PE homeostasis.

Mitochondrial translation deficiency impairs NAD+ -mediated lysosomal acidification.

Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation-deficient hearts from p32-knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD+ ) biosynthetic enzymes-Nmnat3 and Nampt-and NAD+ levels were decreased, suggesting that NAD+ is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α-Nmnat3-mediated NAD+ production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD+ was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD+ content affected by mitochondrial dysfunction is essential for lysosomal maintenance.

BNIP3-dependent mitophagy promotes cytosolic localization of LC3B and metabolic homeostasis in the liver.

Mitophagy formed the basis of the original description of autophagy by Christian de Duve when he demonstrated that GCG (glucagon) induced macroautophagic/autophagic turnover of mitochondria in the liver. However, the molecular basis of liver-specific activation of mitophagy by GCG, or its significance for metabolic stress responses in the liver is not understood. Here we show that BNIP3 is required for GCG-induced mitophagy in the liver through interaction with processed LC3B; an interaction that is also necessary to localize LC3B out of the nucleus to cytosolic mitophagosomes in response to nutrient deprivation. Loss of BNIP3-dependent mitophagy caused excess mitochondria to accumulate in the liver, disrupting metabolic zonation within the liver parenchyma, with expansion of zone 1 metabolism at the expense of zone 3 metabolism. These results identify BNIP3 as a regulator of metabolic homeostasis in the liver through its effect on mitophagy and mitochondrial mass distribution.Abbreviations: ASS1, arginosuccinate synthetase; BNIP3, BCL2/adenovirus E1B interacting protein 3; CV, central vein; GCG - glucagon; GLUL, glutamate- ammonia ligase (glutamine synthetase); HCQ, hydroxychloroquine; LIR, LC3-interacting region; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 beta; mtDNA:nucDNA, ratio of mitochondrial DNA to nuclear DNA; PV, periportal vein; TOMM20, translocase of outer mitochondrial membrane protein 20.

TRMU deficiency: A broad clinical spectrum responsive to cysteine supplementation.

TRMU is a nuclear gene crucial for mitochondrial DNA translation by encoding tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase, which thiolates mitochondrial tRNA. Biallelic pathogenic variants in TRMU are associated with transient infantile liver failure. Other less common presentations such as Leigh syndrome, myopathy, and cardiomyopathy have been reported. Recent studies suggested that provision of exogenous L-cysteine or N-acetylcysteine may ameliorate the effects of disease-causing variants and improve the natural history of the disease. Here, we report six infants with biallelic TRMU variants, including four previously unpublished patients, all treated with exogenous cysteine. We highlight the first report of an affected patient undergoing orthotopic liver transplantation, the long-term effects of cysteine supplementation, and the ability of the initial presentation to mimic multiple inborn errors of metabolism. We propose that TRMU deficiency should be suspected in all children presenting with persistent lactic acidosis and hypoglycemia, and that combined N-acetylcysteine and L-cysteine supplementation should be considered prior to molecular diagnosis, as this is a low-risk approach that may increase survival and mitigate the severity of the disease course.

Functional coupling of presequence processing and degradation in human mitochondria.

The mitochondrial proteome is built and maintained mainly by import of nuclear-encoded precursor proteins. Most of these precursors use N-terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce. As the matrix presequence peptidase PreP has been reported to play a role in Alzheimer's disease, analysis of impaired peptide turnover in human cells is of huge interest. Here, we report the characterization of HEK293T PreP knockout cells. Loss of PreP causes severe defects in oxidative phosphorylation and changes in nuclear expression of stress response marker genes. The mitochondrial defects upon lack of PreP result from the accumulation of presequence peptides that trigger feedback inhibition of MPP and accumulation of nonprocessed precursor proteins. Also, the mitochondrial intermediate peptidase MIP that cleaves eight residues from a subset of precursors after MPP processing is compromised upon loss of PreP suggesting that PreP also degrades MIP generated octapeptides. Investigation of the PrePR183Q patient mutation associated with neurological disorders revealed that the mutation destabilizes the protein making it susceptible to enhanced degradation and aggregation upon heat shock. Taken together, our data reveal a functional coupling between precursor processing by MPP and MIP and presequence degradation by PreP in human mitochondria that is crucial to maintain a functional organellar proteome.

SLC25A51 is a mammalian mitochondrial NAD+ transporter.

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 controversial3-5. 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.