Silent information regulator (Sir) protein, Sir2, is an NAD+-dependent histone deacetylase that mediates chromatin silencing and provides evidence for a genetic link between silencing, metabolism, and aging in yeast. The role of the mammalian Sir2 homolog, Sirt4, is not known. I will analyze the biochemistry and biology of Sirt4. Amino acid sequence comparisons suggest that Sift4 binds NAD+, but may recognize unique substrates. I will develop a protein purification system for Sift4 and measure deacetylase and ADP ribosyltransferase activity. To identify in vivo binding partners and cellular targets, I will combine immunoprecipitation, gel filtration, and yeast 2-hybrid techniques. Does Sirt4 function to regulate cell death or aging in mammals? I will study in vivo function of Sift4 by cell culture siRNA experiments and the creation of Sirt4 null mice. My findings will provide vital information on how Sir2 homologs function in higher organisms.