The yeast Sir2 protein, required for transcriptional silencing, has an NAD ؉ -dependent histone deacetylase (HDA) activity. Yeast extracts contain a NAD ؉ -dependent HDA activity that is eliminated in a yeast strain from which SIR2 and its four homologs have been deleted. This HDA activity is also displayed by purified yeast Sir2p and homologous Archaeal, eubacterial, and human proteins, and depends completely on NAD ؉ in all species tested. The yeast NPT1 gene, encoding an important NAD ؉ synthesis enzyme, is required for rDNA and telomeric silencing and contributes to silencing of the HM loci. Null mutants in this gene have significantly reduced intracellular NAD ؉ concentrations and have phenotypes similar to sir2 null mutants. Surprisingly, yeast from which all five SIR2 homologs have been deleted have relatively normal bulk histone acetylation levels. The evolutionary conservation of this regulated activity suggests that the Sir2 protein family represents a set of effector proteins in an evolutionarily conserved signal transduction pathway that monitors cellular energy and redox states. T ranscriptional silencing is a regulatory mechanism that results in the inactivation of large blocks of chromosomes via an altered chromatin structure. In Saccharomyces cerevisiae, silencing is observed at the HM silent mating type loci (reviewed in ref. 1), telomeres (2), and at the rDNA locus (3, 4). Although a different subset of proteins is required for silencing at each of the three loci, all types of silencing require Sir2p (3, 5). The Sir2 family of proteins is highly conserved and found in Archaea, eubacteria, and metazoa (6-9). A recent study showed that yeast and mouse Sir2p have NAD ϩ -dependent HDA activity on histone peptides specific for Lys-16 of histone H4 (10), an important residue for silencing (11-13). Earlier work had suggested that Sir2p might have HDA activity. Acetylated histones were inefficiently immunoprecipitated from the silent mating type (HM) loci relative to the expressed mating type (MAT) locus, and overexpression of Sir2p led to changes in levels of bulk histone acetylation (14,15). Other recent papers demonstrated a phosphotransferase activity for Sir2p, with NAD ϩ as the source of phosphate and a variety of proteins implicated as targets of ADP ribosylation (9, 16). A sir2 missense mutation that destroys this in vitro activity also destroys silencing in vivo. These results suggest that the Sir2p family is a group of ADP-ribosyl transferases (ARTs).We show here that Archaeal, eubacterial, and human Sir2 proteins, like Sir2p, have potent NAD ϩ -dependent HDA activity in vitro. The importance of NAD ϩ to the in vivo activity of Sir2p is underscored by our finding that mutations in the S. cerevisiae NPT1 gene lead to severe silencing defects. NPT1 encodes a nicotinate phosphoribosyltransferase, required for NAD ϩ synthesis through a salvage pathway. Intracellular NAD ϩ levels are significantly lower in npt1 null mutants than in the wild type, providing independent evidence that NAD ϩ is critic...

Generalized transcriptional repression of large chromosomal regions in Saccharomyces cerevisiae occurs at the silent mating loci and at telomeres and is mediated by the silent information regulator (SIR) genes. We have identified a novel form of transcriptional silencing in S. cerevisiae in the ribosomal DNA (rDNA) tandem array. Tyl retrotransposons marked with a weakened URA3 gene (Tyl-mURAS) efficiently integrated into rDNA. The tnURA3 marker in rDNA was transcriptionally silenced in a SIR2-dependent manner. METIS and LEU2 were also partially silenced, indicating that rDNA silencing may be quite general. Deletion of SIR4 enhanced mURA3 and METIS silencing, but deletion of SIRl or SIRS did not affect silencing, indicating that the mechanism of silencing differs from that at telomeres and silent mating loci. Deletion of SIR2 resulted in increased psoralen cross-linking of the rDNA in vivo, suggesting that a specific chromatin structure in rDNA down-regulates polymerase II promoters.

Although NAD(+) biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD(+) precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD(+) precursor that is converted to nicotinamide mononucleotide by specific nicotinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD(+) synthesis through two pathways, the Nrk1 pathway and the Urh1/Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD(+) metabolism in the absence of nicotinamide-riboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD(+) and increases Sir2 function.

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS-solubilized triads using immunoaffinity chromatography. The purified receptor had a [aH]ryanodine-binding capacity (Bm~,) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +__ 10 pS in 54 mM tram Ca, (c) a long-term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio Pc~/Pxn, ~-14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (3~m~ for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio Pc~/Px ~ 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine.