Abstract
Among the enzymes involved in NAD homeostasis, nicotinamide mononucleotide adenylyltransferases (NMNAT1-3) are central to intracellular NAD formation. Although NMNAT3 is postulated to be a mitochondrial enzyme contributing to NAD-dependent organelle functioning, information on endogenous proteins is lacking. We report that in human cells a single gene nmnat3 localized on chromosome 3 codes for two mRNA splice variants NMNATv1 and FKSG76, whereas the previously reported NMNAT3v2 transcript is not present. However, NMNAT3v1 and FKSG76 proteins are not detectable, consistent with the finding that an upstream ORF in their mRNAs negatively regulates translation. NMNAT3v1 transfection demonstrates that the protein is cytosolic and inactive, whereas FKSG76 is mitochondrial but operates NAD cleavage rather than synthesis. In keeping with the lack of NMNAT3, we show that extracellular NAD, but not its metabolic precursors, sustains mitochondrial NAD pool in an ATP-independent manner. Data of the present study modify the scenario of the origin of mitochondrial NAD by showing that, in human cells, NMNAT3 is absent in mitochondria, and, akin to plants and yeast, cytosolic NAD maintains the mitochondrial NAD pool.
Highlights
During the last several years, we witnessed a renewed interest in the biosynthesis of nicotinamide adenine dinucleotide (NAD) [1,2]
These data taken together indicate that nmnat3 on chromosome 3 codes for a pre-mRNA from which FKSG76 and NMNAT3v1 but not NMNAT3v2 transcripts originate by alternative splicing (Fig. 1E)
The present study sought to investigate the functional relevance of NMNAT3 to mitochondrial NAD homeostasis
Summary
During the last several years, we witnessed a renewed interest in the biosynthesis of nicotinamide adenine dinucleotide (NAD) [1,2] This is due to the understanding that NAD homeostasis is not exclusively based on redox reactions (i.e. mutual conversion of NAD into NADH with conservation of the dinucleotide moiety) and on the irreversible transformation of NAD into an expanding array of metabolites endowed with pleiotypic signaling properties [1,3]. The main pathway of NAD resynthesis is a two-step metabolic route. It stems from the conversion of nicotinamide (Nam) into nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyl-transferase, that is followed by the conversion of NMN into NAD by nicotinamide mononucleotide adenylyltransferase (NMNAT) [5]. While NMNAT1 is a nuclear enzyme responsible for the majority of NAD resynthesis [10], and NMNAT2 is a protein probably docked to the external surface of the endoplasmic reticulum contributing to cytosolic NAD rescue [11], less is known about NMNAT3
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