Telomeric and rDNA Silencing in Saccharomyces cerevisiae Are Dependent on a Nuclear NAD⁺ Salvage Pathway

Corresponding author: Jeffrey S. Smith, Department of Biochemistry and Molecular Genetics, Jordan Hall, Box 800733, Charlottesville, VA 22908-0733., jss5y{at}virginia.edu (E-mail)

Communicating editor: L. PILLUS

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 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⁺.

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