title: Insight into molecular and functional properties of NMNAT3 reveals new hints of NAD homeostasis within human mitochondria.
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.
authors.affiliation: Department of Health Sciences, University of Florence, Florence, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Felici
authors.forename: Andrea
authors.initials: A
authors.lastname: Lapucci
authors.forename: Matteo
authors.initials: M
authors.lastname: Ramazzotti
authors.forename: Alberto
authors.initials: A
authors.lastname: Chiarugi
mesh_terms: D001483:Base Sequence; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D057809:HEK293 Cells; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism* / Q000494:pharmacology; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D020033:Protein Isoforms; D012333:RNA, Messenger; D009243:NAD; C484007:NMNAT1 protein, human; C484008:NMNAT2 protein, human; C484009:NMNAT3 protein, human; D009612:Nicotinamide-Nucleotide Adenylyltransferase