Telomeric and rDNA silencing in Saccharomyces cerevisiae are dependent on a nuclear NAD(+) salvage pathway.
The Sir2 protein is an NAD(+)-dependent protein deacetylase that is required for silencing at the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA). Mutations in the NAD(+) salvage gene NPT1 weaken all three forms of silencing and also cause a reduction in the intracellular NAD(+) level. We now show that mutation of a highly conserved histidine residue in Npt1p results in a silencing defect, indicating that Npt1p enzymatic activity is required for silencing. Deletion of another NAD(+) salvage pathway gene called PNC1 caused a less severe silencing defect and did not significantly reduce the intracellular NAD(+) concentration. However, silencing in the absence of PNC1 was completely dependent on the import of nicotinic acid from the growth medium. Deletion of a gene in the de novo NAD(+) synthesis pathway BNA1 resulted in a significant rDNA silencing defect only on medium deficient in nicotinic acid, an NAD(+) precursor. By immunofluorescence microscopy, Myc-tagged Bna1p was localized throughout the whole cell in an asynchronously growing population. In contrast, Myc-tagged Npt1p was highly concentrated in the nucleus in approximately 40% of the cells, indicating that NAD(+) salvage occurs in the nucleus in a significant fraction of cells. We propose a model in which two components of the NAD(+) salvage pathway, Pnc1p and Npt1p, function together in recycling the nuclear nicotinamide generated by Sir2p deacetylase activity back into NAD(+).
Sandmeier
Joseph J
JJ
Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA. jss5y@virginia.edu
Celic
Ivana
I
Boeke
Jef D
JD
Smith
Jeffrey S
JS
D000595:Amino Acid Sequence; D004275:DNA, Ribosomal / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D020868:Gene Silencing / Q000502:physiology*; D006655:Histone Deacetylases / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D009525:Niacin / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016415:Sequence Alignment; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D056565:Sirtuin 2; D037761:Sirtuins; D016615:Telomere / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism
D004275:DNA, Ribosomal; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D015534:Trans-Activators; D009243:NAD; D009525:Niacin; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2; D037761:Sirtuins; D006655:Histone Deacetylases