MCART1/SLC25A51 is required for mitochondrial NAD transport

Abstract

The nicotinamide adenine dinucleotide (NAD+/NADH) pair is a cofactor in redox reactions and is particularly critical in mitochondria as it connects substrate oxidation by the tricarboxylic acid (TCA) cycle to ATP generation by the electron transport chain (ETC) and oxidative phosphorylation. While a mitochondrial NAD+ transporter has been identified in yeast, how NAD enters mitochondria in metazoans is unknown. Here, we mine gene essentiality data from human cell lines to identify MCART1 (SLC25A51) as co-essential with ETC components. MCART1-null cells have large decreases in TCA cycle flux, mitochondrial respiration, ETC complex I activity, and mitochondrial levels of NAD+ and NADH. Isolated mitochondria from cells lacking or overexpressing MCART1 have greatly decreased or increased NAD uptake in vitro, respectively. Moreover, MCART1 and NDT1, a yeast mitochondrial NAD+ transporter, can functionally complement for each other. Thus, we propose that MCART1 is the long sought mitochondrial transporter for NAD in human cells.

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation

“…Similar to SLC12A8 knockdown, knockdown of NMNAT3 also had no effect on mitochondrial ADP-ribosylation dynamics. These findings strongly support the existence of a separate mitochondrial NAD + transporter ( Davila et al., 2018 ; Girardi et al., 2020 ; Kory et al., 2020 ; Luongo et al., 2020 ) and suggest that both transporters (NMN and NAD + ) are important and necessary for the establishment and regulation of different subcellular NAD + pools.

Figure 4 Lack of NMNAT3 and the NMN Transporter SLC12A8 Extends Nuclear ADP-Ribosylation after H 2 O 2 Treatment (A) Schematic overview of compartmentalized NAD + synthesis and breakdown in most transformed cells.

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“…Our results indicate that restoration of mitochondrial NAD + can be mediated by NMN uptake. Moreover, recent studies identified MCART1/SLC25A51 as the mitochondrial NAD + transporter responsible for direct uptake of NAD + ( Davila et al., 2018 ; Girardi et al., 2020 ; Kory et al., 2020 ). Together, these findings strongly indicate that mitochondria can regulate their NAD + levels via different, partially redundant mechanisms.…”

Epistasis-driven identification of SLC25A51 as a regulator of human mitochondrial NAD import

“…While additional data, including transport assays with reconstituted recombinant protein, will be necessary to definitely prove the role of SLC25A51 as NAD+ transporter and fully define its substrate profile and transport mechanism, the multiple functional links here described convincingly converge on a functional annotation of SLC25A51 as an enabler of NAD+ accumulation in mitochondria. This annotation is corroborated by two recent publications 44,45 providing complementary evidence, including metabolomics analyses and yeast complementation experiments, of SLC25A51 being the longhypothesized mitochondrial NAD+ transporter in human cells. Further studies addressing the interplay between SLC25A51 and the close paralogs SLC25A52, which also showed the ability to transport NAD+ 44,45 , and the orphan SLC25A53 are also expected to shed light on the physiological relevance and regulatory mechanisms of this SLC.…”

“…This annotation is corroborated by two recent publications 44,45 providing complementary evidence, including metabolomics analyses and yeast complementation experiments, of SLC25A51 being the longhypothesized mitochondrial NAD+ transporter in human cells. Further studies addressing the interplay between SLC25A51 and the close paralogs SLC25A52, which also showed the ability to transport NAD+ 44,45 , and the orphan SLC25A53 are also expected to shed light on the physiological relevance and regulatory mechanisms of this SLC. Despite the high degree of functional redundancy displayed by transporters 46 , we show here that informative patterns of genetic interactions can be systematically derived for this family, allowing the generation of testable hypotheses for the de-orphanization of poorly characterized SLCs.…”

Chronic depletion of subcellular NAD pools reveals their interconnectivity and a buffering function of mitochondria

“…A putative buffering function of the mitochondrial NAD + pool would be intrinsically linked to the mechanisms underlying the establishment and maintenance of that pool. Recent studies have shown that the main route for the maintenance of mitochondrial NAD + levels is associated with SLC25A51/MCART1, an NAD + carrier in the mitochondrial inner membrane 49,50 . Therefore, the role of the previously identified mitochondrial NMNAT3 38,82 in mitochondrial NAD + homeostasis is unclear.…”