title: Deletion or overexpression of mitochondrial NAD+ carriers in Saccharomyces cerevisiae alters cellular NAD and ATP contents and affects mitochondrial metabolism and the rate of glycolysis.
abstract: The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy.
authors.forename: Gennaro
authors.initials: G
authors.lastname: Agrimi
authors.forename: Luca
authors.initials: L
authors.lastname: Brambilla
authors.forename: Gianni
authors.initials: G
authors.lastname: Frascotti
authors.forename: Isabella
authors.initials: I
authors.lastname: Pisano
authors.forename: Danilo
authors.initials: D
authors.lastname: Porro
authors.forename: Marina
authors.initials: M
authors.lastname: Vai
authors.forename: Luigi
authors.initials: L
authors.lastname: Palmieri
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D002352:Carrier Proteins; D003470:Culture Media; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D000255:Adenosine Triphosphate; D005947:Glucose