Nicotinamide clearance by Pnc1 directly regulates Sir2-mediated silencing and longevity

Abstract

The Saccharomyces cerevisiae Sir2 protein is an NAD(+)-dependent histone deacetylase (HDAC) that functions in transcriptional silencing and longevity. The NAD(+) salvage pathway protein, Npt1, regulates Sir2-mediated processes by maintaining a sufficiently high intracellular NAD(+) concentration. However, another NAD(+) salvage pathway component, Pnc1, modulates silencing independently of the NAD(+) concentration. Nicotinamide (NAM) is a by-product of the Sir2 deacetylase reaction and is a natural Sir2 inhibitor. Pnc1 is a nicotinamidase that converts NAM to nicotinic acid. Here we show that recombinant Pnc1 stimulates Sir2 HDAC activity in vitro by preventing the accumulation of NAM produced by Sir2. In vivo, telomeric, rDNA, and HM silencing are differentially sensitive to inhibition by NAM. Furthermore, PNC1 overexpression suppresses the inhibitory effect of exogenously added NAM on silencing, life span, and Hst1-mediated transcriptional repression. Finally, we show that stress suppresses the inhibitory effect of NAM through the induction of PNC1 expression. Pnc1, therefore, positively regulates Sir2-mediated silencing and longevity by preventing the accumulation of intracellular NAM during times of stress.

FIG. 1.

Schematic diagram of NAD⁺ biosynthesis and salvage in yeast cells. The de novo synthesis pathway starts with tryptophan (Trp) and is converted to NaMN by the BNA1 through BNA6 gene products. NaMN is then converted to deamido-NAD (NaAD) by the nicotinic acid/NAM mononucleotide adenylyltransferases NMA1 and NMA2. NaAD is then converted to NAD⁺ by NAD⁺ synthetase (QNS1). If NAD⁺ is hydrolyzed into NAM and o-acetyl-ADP ribose (not shown) by Sir2 or an NAD glycohydrolase, then the NAD⁺ salvage pathway converts NAM back into NaMN for reentry to the biosynthesis pathway. The PNC1 gene product is a nicotinamidase that converts NAM to nicotinic acid (Na), which is then converted to NaMN by the nicotinic acid phosphoribosyltransferase, Npt1. Nicotinic acid can also be imported to the cell by the Tna1 nicotinic acid permease. It is at present unclear how NAM is imported into the cell.

FIG. 2.

Stimulation of Sir2 HDAC activity by Pnc1 in vitro. (A) HDAC assays were carried out with a Biomol fluorescent HDAC kit (see Materials and Methods). Each reaction mixture contained 1 μg (11.2 pmol) of GST-Sir2 and 200 μM NAD⁺ and was incubated for 30 min at 30°C. Where indicated, 50 μM NAM and 0.5 μg (17.8 pmol) of recombinant His₆-Pnc1 or C167A mutant Pnc1 was added to the reaction mixture. Activity for the Sir2-only reaction was set at 100%. (B) Sir2 activity measured over time in the absence of exogenously added NAM. The amount of activity at the 0.5-h time point without Pnc1 added was assigned an arbitrary value of 1.0. For later time points, the average fold increase in HDAC activity compared to the 0.5-h time point is plotted. (C) Sir2 activity measured over time in the presence of 7.5 μM NAM. Data are plotted in the same way as in panel B. Error bars represent the average deviation from three independent experiments.

FIG. 3.

Regulation of telomeric silencing by NAM and PNC1. Silencing of a telomeric URA3 reporter gene was tested in WT (JJSy143), npt1Δ (JJSy137), and pnc1Δ (JJSy165) strains. (A) Fivefold serial dilutions of cells were spotted onto SC media or SC media containing 5-FOA. The plates were supplemented with 500 μM or 5 mM NAM where indicated. Two independent colonies for each strain type were tested. (B) WT and pnc1Δ strains were transformed with an empty vector (pRS425), a 2μm SIR2 plasmid (pSB765), or a 2μm PNC1 plasmid (pJOE31). The transformants were plated as fivefold serial dilutions onto SC-Leu or onto SC-Leu+FOA that was supplemented with 500 μM or 5 mM NAM where indicated. Photographs were taken after incubation for 2 days.

FIG. 4.

Regulation of rDNA silencing by NAM and PNC1. (A) Silencing of a Ty1-mURA3 reporter integrated into the NTS1 sequence of the rDNA was tested in WT (JS128), sir2Δ (JS163), npt1Δ (JS587), and pnc1Δ (JS902 and JS903) strains. The control strain (JS122) contains Ty1-mURA3 integrated at a non-rDNA location that is not subjected to silencing. Fivefold serial dilutions of cells were spotted onto SC of SC−Ura medium. The medium was supplemented with 500 μM or 5 mM NAM where indicated. Photographs were taken after incubation for 2 days. (B) WT and pnc1Δ strains were transformed with an empty vector (pRS425), a 2μm SIR2 plasmid (pSB765), or a 2μm PNC1 plasmid (pJOE31). Serial dilutions were spotted onto SC−Leu or SC−Leu−Ura medium. The medium was supplemented with 500 μM or 5 mM NAM where indicated. Photographs were taken after incubation for 3 days.

FIG. 5.

Regulation of HM silencing by NAM and PNC1. (A) Mating assay to measure the silencing of the HMR and HML loci in general. WT (DSY50), pnc1Δ (DSY46), and npt1Δ (DSY75) strains were mated to a MATa tester strain (SY35) in the presence of 0, 2.5, or 5 mM NAM for 4 h. Fivefold serial dilutions of cells were spotted. Growth of the resulting diploids on SD plates is indicative of silencing. The MATa tester strain was spotted as a negative control. (B) Silencing at HMR was measured by using WT or sir2Δ versions of a hmrΔA::TRP1 reporter strain that were transformed with either a pRS425 empty vector or the high-copy-number PNC1 vector, pJOE31.

FIG. 6.

NAM and PNC1 regulate longevity. The replicative life span of strains containing either an empty CEN/ARS pRS415 vector (DSY67) or a CEN/ARS PNC1 pJOE54 plasmid (DSY68) was tested on rich YPD growth medium. Where indicated, 5 mM NAM was added. The data are plotted as the percentage of mother cells still viable (y axis) after each successive generation (x axis).

FIG. 7.

Stress-mediated silencing regulation by PNC1. (A) Northern blot analysis of steady-state PNC1 RNA levels from cells grown normally (at 30°C), at an elevated temperature (37°C), or in the presence of 0.02% MMS. The strains contained either an empty vector (pRS425) or the 2μm PNC1 plasmid (pJOE31). The ACT1 gene was used as a loading control. (B) Telomeric silencing spot assay for silencing of a URA3 reporter. WT (JJSy143), npt1Δ (JJSy137), and pnc1Δ (JJSy165) strains were spotted onto SC−Leu medium as a growth control, and SC−Leu+FOA medium to measure silencing. To partially inhibit silencing, 1 mM NAM was added. The strains were grown either at 30 or 37°C or in the presence of 0.02% MMS.