| As knowledge of the diverse roles played by the Sir2 family of NAD +-dependent protein deacetylases expands, it becomes increasingly important to understand the mechanisms regulating their catalytic activity, substrate specificity and localization. This dissertation explores the regulation of Hst2, a cytoplasmic sirtuin in budding yeast that influences the epigenetic silencing of nuclear genes. Hst2's cellular function remains an intriguing open question in light of its robust deacetylase activity, cytoplasmic localization and lack of autonomous mutant phenotypes. With the goal of elucidating the function and regulation of Hst2, this study presents an in depth characterization of the genetic, biochemical and cell biological behavior of HST2 and its gene product in budding yeast. Through a chromatographic analysis of Hst2 derived from yeast lysate, it is determined that Hst2 exists primarily in a monomeric form in vivo, although a higher mass, minor species is also present. A single hst2Delta mutant phenotype is presented, as well as genetic characterizations of several truncation alleles of HST2 based on crystallographic data. The region encoding the carboxy-terminal extension of Hst2 is determined to be particularly important for Hst2 function. Functional interactions between HST2 and NAD+ synthesis genes are also explored. Lastly, it is determined that the apparent cytoplasmic localization of Hst2 is due to efficient, facilitated nuclear export. An HST2 mutant deficient in export displayed dominant, dosage-dependent phenotypes that suggest a physiological role for Hst2 in the nucleus. Potential functions and modes of regulation for Hst2 are discussed in the final chapter. |
